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Amyloidosis is a localized or systemic process where extracellular insoluble plasma protein fibers are deposited into tissues. Localized amyloidosis is rare and curable by surgical resection. While the head and neck region represents 19% of localized amyloidosis cases, only one other case of bilateral involvement of the pharyngeal tonsils has been published in the international literature. We report a case of asymptomatic amyloidosis isolated to the bilateral palatine tonsils and a cervical lymph node in a 59-year-old male. Systemic amyloidosis was ruled out through multidisciplinary consultation, and resection of the masses was performed. This represents the second reported case of bilateral tonsillar amyloidosis.

The increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage. Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay. Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage. There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately.

Microcystins are common freshwater cyanobacterial toxins that affect liver function. The toxicities of five microcystin congeners (microcystin-LA (MCLA), MCLR, MCLY, MCRR, and MCYR) commonly observed in harmful algal blooms (HABs) were evaluated in BALB/c mice after a single oral administration of doses ranging from those that were no observed adverse effect levels (NOAELs) to lowest observed adverse effect levels (LOAELs). Animals were monitored for changes in behavior and appearance, and euthanized 24 h after dosing. Test endpoints included clinical changes, necropsy observations, and serum indicators of hepatic toxicity and general homeostasis. Doses were 0.5&ndash, 7 mg/kg MCLA, 0.5&ndash, 11 mg/kg MCLR, 1&ndash, 7 mg/kg MCLY, 7&ndash, 22 mg/kg MCRR, and 3&ndash, 11 mg/kg MCYR. MCLA at 3 mg/kg elevated liver/body weight ratio and liver score, ALT, AST, and GLDH, indicating hepatic toxicity, reduced serum glucose and highly elevated total serum bilirubin. MCLR and MCLY induced similar effects with LOAELs of 5 mg/kg, although a greater extent and severity of effects were observed in MCLR animals. MCRR exposure at 22 mg/kg was associated with reduced serum glucose. MCYR induced scattered liver effects at 7 mg/kg and reduced serum glucose levels at 5 mg/kg. The results indicate significant differences in congener-induced toxicity after microcystin exposure.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,1,2,2-tetrafluoroethoxy)propan-2-yl]oxyethane-1-sulfonic acid (PFESA-BP2) was first detected in 2012 in the Cape Fear River downstream of an industrial manufacturing facility. It was later detected in the finished drinking water of municipalities using the Cape Fear River for their water supply. No toxicology data exist for this contaminant despite known human exposure. To address this data gap, mice were dosed with PFESA-BP2 at 0, 0.04, 0.4, 3, and 6 mg/kg-day for 7 days by oral gavage. As an investigative study, the final dose groups evolved from an original dose of 3 mg/kg which produced liver enlargement and elevated liver enzymes. The dose range was extended to explore a no effect level. PFESA-BP2 was detected in the sera and liver of all treated mice. Treatment with PFESA-BP2 significantly increased the size of the liver for all mice at 3 and 6 mg/kg-day. At the 6 mg/kg-day dose, the liver more than doubled in size compared to the control group. Male mice treated with 3 and 6 mg/kg-day and females treated with 6 mg/kg-day demonstrated significantly elevated serum markers of liver injury including alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), and liver/body weight percent. The percent of PFESA-BP2 in serum relative to the amount administered was similar in male and female mice, ranged from 9 to 13 %, and was not related to dose. The percent accumulation in the liver of the mice varied by sex (higher in males), ranged from 30 to 65 %, and correlated positively with increasing dose level.

Microcystins (MCs) are common cyanobacterial toxins that occur in freshwaters worldwide. Only two of the &gt, 200 MC variants have been tested for potential toxicity after oral exposure. This paper reports on the toxicity of 10 different MC congeners identified in algal blooms, microcystin-LR (MCLR), MCLA, MCLF, MCLW, MCLY, MCRR, [Asp3]MCRR, [Asp3,Dhb7]MCRR, MCWR, and MCYR after single administrations to BALB/c mice. In a preliminary MCLR dose&ndash, response study of 3 to 9 mg/kg doses, &ge, 5 mg/kg induced clinical changes, increased serum levels of ALT, AST, and GLDH, liver congestion, increased liver/body weight ratios, and reduced serum glucose and total protein. Based on the extent of these effects, the 10 congeners were administered as single 7 mg/kg oral doses and toxicity evaluated. The greatest toxicity was observed with MCLA and MCLR including a high percentage of moribundity. In addition to eliciting effects similar to those listed above for MCLR, MCLA also induced serum alterations indicative of jaundice. MCLY, and MCYR induced changes like those noted with MCLR, but to lesser extents. MCLW and MCLF exhibited some serum and morphological changes associated with hepatic toxicity, while there were few indications of toxicity after exposures to MCRR, [Asp3]MCRR, [Asp3,Dhb7]MCRR, or MCWR. These data illustrate a wide spectrum of hepatic effects and different potencies of these MC congeners.

Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre‐activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor‐infiltrating CD56+ NK cells in formalin‐fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin‐based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3–4) showed significantly decreased overall survival (OS; p = 0.008), local progression‐free survival (LPFS; p = 0.034) and distant metastases‐free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0–2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16‐ (p = 0.001), p53‐ (p = 0.0003) and HPV16 DNA‐negative (p = 0.001) tumors. The absence or low numbers of tumor‐infiltrating CD56+ NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor‐infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor‐infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers., What's new? It's difficult to predict how a patient with squamous‐cell carcinoma of the head and neck (SCCHN) will respond to treatment, because every tumor is different. In this study, the authors identified two pre‐treatment measures that were associated with poor prognosis following surgery and RCT: high levels of staining for a protein called Hsp70 in tumor cells, and low numbers of tumor‐infiltrating NK lymphocytes. These measures may thus serve as useful prognostic biomarkers for predicting the response of SCCHN to therapy.

Background Asymptomatic carotid artery stenosis (ACS) has a low risk of stroke. To achieve an advantage over noninterventional best medical treatment (BMT), carotid endarterectomy (CEA) or carotid artery stenting (CAS) must be performed with the lowest possible risk of stroke. Therefore, an analysis of risk-elevating factors is essential. Grade of ipsilateral and contralateral stenosis as well as plaque morphology are known risk factors in ACS. Methods The randomized, controlled, multicenter SPACE-2 trial had to be stopped prematurely after recruiting 513 patients. 203 patients were randomized to CEA, 197 to CAS, and 113 to BMT. Within one year, risk factors such as grade of stenosis and plaque morphology were analyzed. Results Grade of contralateral stenosis (GCS) was higher in patients with any stroke (50%ECST vs. 20%ECST; p=0.012). Echolucent plaque morphology was associated with any stroke on the day of intervention (OR 5.23; p=0.041). In the periprocedural period, any stroke was correlated with GCS in the CEA group (70%ECST vs. 20%ECST; p=0.026) and with echolucent plaque morphology in the CAS group (6% vs. 1%; p=0.048). In multivariate analysis, occlusion of the contralateral carotid artery (CCO) was associated with risk of any stroke (OR 7.00; p=0.006), without heterogeneity between CEA and CAS. Conclusion In patients with asymptomatic carotid artery stenosis, GCS, CCO, as well as echolucent plaque morphology were associated with a higher risk of cerebrovascular events. The risk of stroke in the periprocedural period was increased by GCS in CEA and by echolucent plaque in CAS. Due to small sample size, results must be interpreted carefully.

Radiotherapy (RT) is an established treatment for patients with primary and recurrent prostate cancer. Herein, the effects of definitive and salvage RT on the composition of lymphocyte subpopulations were investigated in patients with prostate cancer to study potential immune effects. A total of 33 prostate cancer patients were treated with definitive (n = 10) or salvage RT (n = 23) after biochemical relapse. The absolute number of lymphocytes and the distribution of lymphocyte subpopulations were analyzed by multiparameter flow cytometry before RT, at the end of RT, and in the follow-up period. Absolute lymphocyte counts decreased significantly after RT in both patient groups and a significant drop was observed in the percentage of B cells directly after RT from 10.1 ± 1.3 to 6.0 ± 0.7% in patients with definitive RT and from 9.2 ± 0.8 to 5.8 ± 0.7% in patients with salvage RT. In contrast, the percentages of T and natural killer (NK) cells remained unaltered directly after RT in both patient groups. However, 1 year after RT, the percentage of CD3+ T cells was significantly lower in patients with definitive and salvage RT. The percentage of regulatory T cells was slightly upregulated in primary prostate cancer patients after definitive RT, but not after salvage RT. Definitive and salvage RT exert similar effects on the composition of lymphocyte subpopulations in prostate cancer patients. Total lymphocyte counts are lower in both patient groups compared to healthy controls and further decreased after RT. B cells are more sensitive to definitive and salvage RT than T and NK cells.

Epidemiological and experimental data suggest that obesity exacerbates the health effects of air pollutants such as ozone (O3). Maternal inactivity and calorically rich diets lead to offspring that show signs of obesity. Exacerbated O3 susceptibility of offspring could thus be manifested by maternal obesity. Thirty-day-old female Long-Evans rats were fed a control (CD) or high-fat (HF) (60% calories) diet for 6 wks and then bred. GD1 rats were then housed with a running wheel (RW) or without a wheel (SED) until parturition, creating four groups of offspring: CD-SED, CD-RW, HF-SED and HF-RW. HF diet was terminated at PND 35 and all offspring were placed on CD. Body weight and %fat of dams were greatest in order; HF-SED > HF-RW > CD-SED > CD-RW. Adult offspring were exposed to O3 for two consecutive days (0.8 ppm, 4 h/day). Glucose tolerance tests (GTT), ventilatory parameters (plethysmography), and bronchoalveolar fluid (BALF) cell counts and protein biomarkers were performed to assess response to O3. Exercise and diet altered body weight and %fat of young offspring. GTT, ventilation and BALF cell counts were exacerbated by O3 with responses markedly exacerbated in males. HF diet and O3 led to significant exacerbation of several BALF parameters: total cell count, neutrophils and lymphocytes were increased in male HF-SED versus CD-SED. Males were hyperglycemic after O3 exposure and exhibited exacerbated GTT responses. Ventilatory dysfunction was also exacerbated in males. Maternal exercise had minimal effects on O3 response. The results of this exploratory study suggest a link between maternal obesity and susceptibility to O3 in their adult offspring in a sex-specific manner.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.

Per- and polyfluoroalkyl substances (PFAS) are organic chemicals with wide industrial and consumer uses. They are found ubiquitously at low levels in the environment and are detectable in humans and wildlife. Perfluorobutane Sulfonate (PFBS) is a short-chained PFAS used to replace perfluorooctane sulfonate in commerce. In general, the rate of clearance for the short-chained PFAS is faster than that for the long-chained congeners. This study evaluated the pharmacokinetic properties of PFBS and its hepatic transcriptional responses in CD-1 mice. Males and females were given PFBS by oral gavage at 30 or 300 mg/kg; controls received 0.5 % Tween-20 vehicle. Trunk blood was collected at 0.5, 1, 2, 4, 8, 16 and 24 h thereafter; liver and kidney were also harvested. Serum and tissue concentrations of PFBS were determined by HPLC-MS-MS. Expression of several hepatic nuclear receptor target genes was determined by qPCR. The half-life of PFBS was estimated as 5.8 h in the males and 4.5 h in the females. Tmax was reached within 1−2 h. Volume of distribution was similar between the two sexes (0.32−0.40 L/kg). The rate of PFBS clearance was linear with exposure doses. Within 24 h, serum PFBS declined to less than 5 % of Cmax. PFBS was detected in liver or kidney, although tissue levels of the chemical were only a fraction of those in serum. At 24 h after administration of 300 mg/kg PFBS, elevated expression of several hepatic genes targeted for PPARα, PPARy, and PXR but not by AhR, LXR or CAR was observed, with responses indistinguishable between males and females. Little to no transcriptional response was seen with the 30 mg/kg dose. The short serum half-lives of PFBS (4−5 h) in mice were comparable to those reported in rats. Although detection of PFBS in liver was low compared to that in serum even at the 300 mg/kg dose, the tissue level was sufficient to activate several hepatic nuclear receptors, which may represent an acute response to the chemical at a high dose.

Cylindrospermopsin (CYN) is a toxin associated with numerous species of freshwater cyanobacteria throughout the world. It is postulated to have caused an episode of serious illnesses in Australia through treated drinking water, as well as lethal effects in livestock exposed to water from farm ponds. Toxicity included effects indicative of both hepatic and renal dysfunction. In humans, symptoms progressed from initial hepatomegaly, vomiting, and malaise to acidosis and hypokalemia, bloody diarrhea, and hyperemia in mucous membranes. Laboratory animal studies predominantly involved the intraperitoneal (i.p.) route of administration and confirmed this pattern of toxicity with changes in liver enzyme activities and histopathology consistent with hepatic injury and adverse renal effects. The aim of this study was designed to assess subchronic oral exposure (90 d) of purified CYN from 75 to 300 µg/kg/d in mouse. At the end of the dosing period, examinations of animals noted (1) elevated organ to body weight ratios of liver and kidney at all dose levels, (2) treatment-related increases in serum alanine aminotransferase (ALT) activity, (3) decreased blood urea nitrogen (BUN) and cholesterol concentrations in males, and (4) elevated monocyte counts in both genders. Histopathological alterations included hepatocellular hypertrophy and cord disruption in the liver, as well as renal cellular hypertrophy, tubule dilation, and cortical tubule lesions that were more prominent in males. A series of genes were differentially expressed including Bax (apoptosis), Rpl6 (tissue regeneration), Fabp4 (fatty acid metabolism), and Proc (blood coagulation). Males were more sensitive to many renal end points suggestive of toxicity. At the end of exposure, toxicity was noted at all dose levels, and the 75 µg/kg group exhibited significant effects in liver and kidney/body weight ratios, reduced BUN, increased serum monocytes, and multiple signs of histopathology indicating that a no-observed-adverse-effect level could not be determined for any dose level.

Background and Purpose— In patients who present with acute ischemic stroke while on treatment with non–vitamin K antagonist oral anticoagulants (NOACs), coagulation testing is necessary to confirm the eligibility for thrombolytic therapy. We evaluated the current use of coagulation testing in routine clinical practice in patients who were on NOAC treatment at the time of acute ischemic stroke. Methods— Prospective multicenter observational RASUNOA registry (Registry of Acute Stroke Under New Oral Anticoagulants; February 2012–2015). Results of locally performed nonspecific (international normalized ratio, activated partial thromboplastin time, and thrombin time) and specific (antifactor Xa tests, hemoclot assay) coagulation tests were documented. The implications of test results for thrombolysis decision-making were explored. Results— In the 290 patients enrolled, nonspecific coagulation tests were performed in ≥95% and specific coagulation tests in 26.9% of patients. Normal values of activated partial thromboplastin time and international normalized ratio did not reliably rule out peak drug levels at the time of the diagnostic tests (false-negative rates 11%–44% [95% confidence interval 1%–69%]). Twelve percent of patients apparently failed to take the prescribed NOAC prior to the acute event. Only 5.7% (9/159) of patients in the 4.5-hour time window received thrombolysis, and NOAC treatment was documented as main reason for not administering thrombolysis in 52.7% (79/150) of patients. Conclusions— NOAC treatment currently poses a significant barrier to thrombolysis in ischemic stroke. Because nonspecific coagulation test results within normal range have a high false-negative rate for detection of relevant drug concentrations, rapid drug-specific tests for thrombolysis decision-making should be established. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT01850797.

Oxidative stress (OS) contributes to the neurological and cardio/pulmonary effects caused by adverse metabolic states and air pollutants such as ozone (O3). This study explores the interactive effects of O3 and diet (high-fructose (FRUC) or high&ndash, fat (FAT)) on OS in different rat brain regions. In acute exposure, there was a decrease in markers of reactive oxygen species (ROS) production in some brain regions by diet and not by O3. Total antioxidant substances (TAS) were increased in the cerebellum (CER) and frontal cortex (FC) and decreased in the striatum (STR) by both diets irrespective of O3 exposure. Protein carbonyls (PC) and total aconitase decreased in some brain regions irrespective of exposure. Following subacute exposure, an increase in markers of ROS was observed in both diet groups. TAS was increased in the FC (FAT only) and there was a clear O3 effect where TAS was increased in the FC and STR. Diet increased PC formation within the CER in the FAT group, while the hippocampus showed a decrease in PC after O3 exposure in controls. In general, these results indicate that diet/O3 did not have a global effect on brain OS parameters, but showed some brain region- and OS parameter-specific effects by diets.

The induction of chromosome aberrations in human lymphocytes irradiated in vitro with slow neutrons was examined to assess the maximum low-dose RBE (RBE(M)) relative to (60)Co γ-rays. For the blood irradiations, cold neutron beam available at the prompt gamma activation analysis facility at the Munich research reactor FRM II was used. The given flux of cold neutrons can be converted into a thermally equivalent one. Since blood was taken from the same donor whose blood had been used for previous irradiation experiments using widely varying neutron energies, the greatest possible accuracy was available for such an estimation of the RBE(M) avoiding the inter-individual variations or differences in methodology usually associated with inter-laboratory comparisons. The magnitude of the coefficient α of the linear dose-response relationship (α = 0.400 ± 0.018 Gy(-1)) and the derived RBE(M) of 36.4 ± 13.3 obtained for the production of dicentrics by thermal neutrons confirm our earlier observations of a strong decrease in α and RBE(M) with decreasing neutron energy lower than 0.385 MeV (RBE(M) = 94.4 ± 38.9). The magnitude of the presently estimated RBE(M) of thermal neutrons is-with some restrictions-not significantly different to previously reported RBE(M) values of two laboratories.

The aim of this study was to investigate the effectiveness of different shielding materials in protective clothing using dicentric frequency in human peripheral lymphocytes as a marker of radiation-induced damage. Blood samples from a healthy donor were exposed to 70 kV x-rays behind shielding materials lead (Pb), tin/antimony (Sn + Sb) and bismuth barrier/tin/tungsten (Bi + Sn + W) with the same nominal lead equivalent value of 0.35 mm lead. Irradiation was performed either in contact (exposure position A, containing secondary radiation) or at a distance of 19 cm behind the shielding materials (exposure position B, containing only the unaffected transmitted photons). Using shielding material Sn + Sb, a significantly higher dicentric yield was determined at exposure position A relative to position B, whereas no significant differences were found between the exposure positions using shielding materials Pb or Bi + Sn + W. For doses up to 434.4 mGy at exposure position A, the slopes of the linear dose-response curves for dicentrics obtained behind shielding materials Pb and Bi + Sn + W were not significantly different, whereas a significantly higher slope was determined behind Sn + Sb relative to Pb and Bi + Sn + W. Using moderately filtered 220 kV x-rays as a reference, maximum RBE values at low doses (RBE(M)) of 1.22 ± 0.10, 2.28 ± 0.19 and 1.03 ± 0.12 were estimated immediately behind shielding materials Pb, Sn + Sb and Bi + Sn + W, respectively. These findings indicate a significantly higher RBE(M) of 70 kV x-rays behind shielding material Sn + Sb with respect to Pb or Bi + Sn + W. Using previous dicentric data obtained for exposure of blood from the same donor to x-rays at energies lower than 70 kV, it can be assumed that the increased RBE(M) of the broad spectrum of 70 kV x-rays (mean energy of 44.1 keV) may be attributed predominately to secondary (mainly fluorescence) radiation generated in the shielding material Sn + Sb that is able to leave the shielding material. Even if it is uncertain whether the marked dependency of the RBE at low doses on photon energy for chromosome aberrations is also representative for late radiation effects in healthy subjects, it should be taken into account that several prospective cohort studies have shown positive associations between higher chromosome aberrations in lymphocytes of healthy subjects and increased cancer incidence. Thus, it can be concluded that any additional biological damage by radiation exposure of healthy subjects, e.g. by using certain non-lead based shielding materials of protective clothing, should be avoided.

The relevance of preconceptional and prenatal toxicant exposures for genomic stability in offspring is difficult to analyze in human populations, because gestational exposures usually cannot be separated from preconceptional exposures. To analyze the roles of exposures during gestation and conception on genomic stability in the offspring, stability was assessed via the Comet assay and highly sensitive, semiautomated confocal laser scans of gammaH2AX foci in cord, maternal, and paternal blood as well as spermatozoa from 39 families in Crete, Greece, and the United Kingdom. With use of multivariate linear regression analysis with backward selection, preconceptional paternal smoking (% tail DNA: P>0.032; gammaH2AX foci: P>0.018) and gestational maternal (% tail DNA: P>0.033) smoking were found to statistically significantly predict DNA damage in the cord blood of F1 offspring. Maternal passive smoke exposure was not identified as a predictor of DNA damage in cord blood, indicating that the effect of paternal smoking may be transmitted via the spermatozoal genome. Taken together, these studies reveal a role for cigarette smoke in the induction of DNA alterations in human F1 offspring via exposures of the fetus in utero or the paternal germline. Moreover, the identification of transgenerational DNA alterations in the unexposed F1 offspring of smoking-exposed fathers supports the claim that cigarette smoke is a human germ cell mutagen.-Laubenthal, J., Zlobinskaya, O., Poterlowicz, K., Baumgartner, A., Gdula, M. R., Fthenou, E., Keramarou, M., Hepworth, S. J., Kleinjans, J. C. S., van Schooten, F.-J., Brunborg, G., Godschalk, R. W., Schmid, T. E., Anderson, D. Cigarette smoke-induced transgenerational alterations in genome stability in cord blood of human F1 offspring.

The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet revealed that propiconazole- and triadimefon-induced mutations do not represent general clonal expansion of background mutations, and support the hypothesis that they arise from the accumulation of endogenous reactive metabolic intermediates within the liver in vivo. We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies to determine if there were quantitative or qualitative differences between mice receiving tumorigenic or nontumorigenic conazoles compared to concurrent control animals. We resolved and quantitated 16 individual adduct spots by (32)P postlabelling and thin layer chromatography using three solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. This same group of endogenous adducts were significantly correlated with mutant frequency across all treatment groups (P = 0.002), as were total endogenous DNA adduct levels (P = 0.005). We hypothesise that this treatment-related increase in endogenous DNA adducts, together with concomitant increases in cell proliferation previously reported to be induced by conazoles, explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon.

To examine if the sequential treatment of Avastin and Macugen is safe and more efficient than the mono-therapies in a prospective randomized masked pilot study.Subjects with exudative age-related macular degeneration were randomized to receive three intravitreal injections of either 1 mg of Avastin, 0.3 mg Macugen, or first 1 mg Avastin followed by retreatment of 0.3 mg Macugen. Follow-up examinations were performed after 1, 6, 12 weeks, and 6 months.Forty-eight subjects were included (13:18:17). Avastin resulted in lasting significant changes in visual acuity at 6 weeks, increase in contrast sensitivity at 6 weeks, and a significant decrease in macular thickness after 6 and 12 weeks. Macugen showed a significant decrease in retinal thickness after 6 weeks, but a significant decrease in visual acuity after 6 months, and a significant decrease in contrast sensitivity after 12 weeks and 6 months. The sequential treatment showed a decrease in retinal thickness after 1 and 12 weeks.Avastin alone is more effective in increasing visual acuity and contrast sensitivity and decreasing retinal thickness, than Macugen or the sequential treatment. We conclude that the sequential treatment of Avastin with Macugen is safe, but the single treatment of Avastin is more efficient.

Background Benzene is a common industrial chemical known to induce leukemia and other blood disorders, as well as aneuploidy, in both human blood cells and sperm at exposures > 10 ppm. Recent reports have identified health effects at exposure levels < 1 ppm, the permissible exposure limit (PEL; 8 hr) set by the U.S. Occupational Safety and Health Administration. Objective We investigated whether occupational exposures to benzene near 1 ppm induce aneuploidy in sperm. Methods We used multicolor fluorescence in situ hybridization to measure the incidence of sperm with numerical abnormalities of chromosomes X, Y, and 21 among 33 benzene-exposed men and 33 unexposed men from Chinese factories. Individual exposures were assessed using personal air monitoring and urinary concentrations of benzene and trans,trans-muconic acid (E,E-MA). Air benzene concentrations were not detectable in unexposed men; in exposed men, concentrations ranged from below the detection limit to 24 ppm (median, 2.9 ppm), with 27% of exposed men (n = 9) having concentrations of ≤ 1 ppm. Exposed men were categorized into low and high groups based on urinary E,E-MA (median concentrations of 1.9 and 14.4 mg/L, respectively; median air benzene of 1 and 7.7 ppm, respectively), and aneuploidy frequencies were compared with those of unexposed men. Results Sperm aneuploidy increased across low- and high-exposed groups for disomy X [incidence rate ratio (IRR) = 2.0; 95% confidence interval (CI), 1.1–3.4; and IRR = 2.8; 95% CI, 1.5–4.9, respectively], and for overall hyperhaploidy for the three chromosomes investigated (IRR = 1.6; 95% CI, 1.0–2.4; and IRR = 2.3; 95% CI, 1.5–3.6, respectively). We also found elevated disomy X and hyperhaploidy in the nine men exposed to ≤ 1 ppm benzene compared with unexposed men (IRR = 1.8; 95% CI, 1.1–3.0; and IRR = 2.0; 95% CI, 1.1–3.9, respectively). Conclusions Benzene appeared to increase the frequencies of aneuploid sperm for chromosomes associated with chromosomal abnormality syndromes in human offspring, even in men whose air benzene exposure was at or below the U.S. permissible exposure limit.

To compare possible toxic effects of membrane blue and infracyanine green used as vital stains in macular surgery. Vital stains are used in vitreoretinal surgery to perform peeling of the internal limiting membrane and idiopathic epiretinal membrane. There are many controversial studies about their toxicity, safety, and their effects on the retinal pigment epithelium and the neuroretinal elements. To compare possible toxicities of the two vital stains membrane blue and infracyanine green in vivo, we conducted a prospective, randomized, clinical trial.A prospective, randomized clinical trial including 30 eyes of 30 patients with either full-thickness macular hole or idiopathic epiretinal membrane were included and randomized 1:1 to receive either membrane blue or infracyanine green during vitreoretinal surgery. Complete ophthalmic examinations, including optical coherence tomography and peripheral visual field were performed preoperatively, and 1, 3, and 6 months postoperatively in our clinic. The main outcome measure was the peripheral visual field. Data was analysed with Student's t-test and Pearson's correlation coefficient.Three months after surgery there was a significant difference in increase in visual field in the superior region in favor of the membrane blue group (p = 0.021). Eight eyes (53%) of the infracyanine group had a decrease in temporal visual fields of at least 5 degrees .Although there was a significant difference in visual fields between the groups after 3 months in the superior region, there were no more significant differences between the two groups after 6 months. However, due to the decrease in the temporal visual field in some eyes, we conclude that membrane blue is less toxic.

Purpose: Melatonin (N-acetyl-5-methoxytryptamine) (MT) is a hormone that acts as an antioxidant. It is produced by the pineal gland and within the retina; its release is blocked by light entering the eye. We examined whether MT daytime levels differ between pseudophakic patients with age-related macular degeneration (ARMD) and pseudophakic subjects without any ocular pathology of the same age. Methods: A prospective, cross-sectional, observational study was performed. Pseudophakic patients of the same age group were included. Patients underwent complete ophthalmic examinations and blood sampling between 08:00 and 10:00 hr. MT daytime value in the serum was the main outcome measure. Results: Sixty-nine pseudophakic patients were included. Fifty patients with exudative and non-exudative ARMD were in the study group while 19 patients were controls. Patients with ARMD had significantly higher daytime levels of MT (P = 0.003). There were significant differences in MT daytime levels between the exudative and non-exudative forms (P = 0.009). MT values also correlated with the best-corrected visual acuity (r = )0.285, P = 0.019). Conclusion: These data indicate that pseudophakic patients with ARMD produce more MT during the day compared to pseudophakic subjects without ARMD. This may be caused by the reduced visual acuity in patients with ARMD, whereby less light reaches the photoreceptors, allowing MT secretion to continue during the day. Because MT also acts as an antioxidant and daytime levels are higher in patients with ARMD, these results might be interpreted as a rescue factor.

To investigate the association of the complement factor H gene (CFH)Y402H polymorphism and age-related macular degeneration (AMD) in the Austrian population (Caucasoid descent), and to determine whether there is an association between exposure to Chlamydia pneumoniae-responsible for up to 20% of community-acquired pneumoniae-and the AMD-associated CFHrisk polymorphism.Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis in 75 unrelated AMD patients and compared with 75 healthy, age-matched control subjects. C. pneumoniaeserum IgG was tested by ELISA (RD) in both groups. The association between the CFHY402H genetic polymorphism and the disease was examined by chi (2)-test and logistic regression.CFH Y402H genotypefrequencies differed significantly between AMD patients and healthy controls (1277 TT, 22.7%; 1277 TC, 53.3%; and 1277 CC, 22.7% in the AMD group; 1277 TT, 48.0%; 1277 TC, 38.7%; and 1277 CC, 13.3% in the control group) showing a P-value0.005 (OR:2.920/3.811).No association was found between a positive C. pneumoniae titre and AMD (P=0.192), nor was any association found between C. pneumoniae and the CFH Y402H polymorphism.Our data confirm that the CFHY402H polymorphism is a risk factor for AMD in the Austrian population with a higher frequency of the Y402 polymorphism in AMD patients. No association between preceding C. pneumoniaeinfection and diagnosed AMD was found.

Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear which procedures alter results. In addition, characterization of interindividual and sex-based variation in gene expression is needed to understand sources and extent of variability. Methods: Whole blood was obtained from adult male and female volunteers (n = 42) and stored at various temperatures for various lengths of time. RNA was isolated and RNA quality analyzed. Affymetrix GeneChips (n = 23) were used to characterize gene expression profiles (GEPs) and to determine the effects on GEP of storage conditions, extraction techniques, types of GeneChip, or donor sex. Hierarchical clustering and principal component analysis were used to assess interindividual differences. Regression analysis was used to assess the relative impact of the studied variables. Results: Storage of blood samples for >1 week at 4 °C diminished subsequent RNA quality. Interindividual GEP differences were seen, but larger effects were observed related to RNA extraction technique, GeneChip, and donor sex. The relative importance of the variables was as follows: storage < genechip < extraction technique < donor sex. Conclusion: Sample storage and extraction methods and interindividual differences, particularly donor sex, affect GEP of human whole blood.

Background Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood. Objectives Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far. Methods and results Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b. Conclusions The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.

Triazole fungicides associated with a range of reported male reproductive effects in experimental animals were selected to assess potential toxic modes of action. Wistar Han rats were fed myclobutanil (M: 100, 500, or 2000 ppm), propiconazole (P: 100, 500, or 2500 ppm), or triadimefon (T: 100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 120. One male per litter was necropsied on PND1, 22, 50, or 92. Measurements included anogenital distance (AGD) at PND0, body and organ weights, serum hormone levels, age at preputial separation (PPS), sperm morphology and motility, and fertility and fecundity. AGD was increased by the high dose of all three triazoles, indicating hypervirilization. Triadimefon delayed PPS, consistent with delayed puberty, at 1800 ppm. Relative liver weights were increased at PND1, 50, and 92 by all three triazoles. Hepatocellular hypertrophy was present at PND50 from propiconazole and triadimefon and at PND92 from all three high-dose triazole treatments. Relative pituitary weights were decreased at PND92 by middle- and high-dose myclobutanil treatment. Absolute testis weights were increased at PND1 by myclobutanil, at PND22 by myclobutanil and triadimefon, and at PND50 by propiconazole and triadimefon treatment. Relative ventral prostate weights were increased at PND92 by myclobutanil and triadimefon treatment. Serum testosterone was increased at PND50 by triadimefon and at PND92/99 by all three triazole treatments. Insemination and fertility were impaired by myclobutanil and triadimefon treatment. In addition to the reproductive system effects, total serum thyroxine levels were decreased at PND92 by high-dose triadimefon. These reproductive effects are consistent with the disruption of testosterone homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity.

Four triazole fungicides were studied using toxicogenomic techniques to identify potential mechanisms of action. Adult male Sprague-Dawley rats were dosed for 14 days by gavage with fluconazole, myclobutanil, propiconazole, or triadimefon. Following exposure, serum was collected for hormone measurements, and liver and testes were collected for histology, enzyme biochemistry, or gene expression profiling. Body and testis weights were unaffected, but liver weights were significantly increased by all four triazoles, and hepatocytes exhibited centrilobular hypertrophy. Myclobutanil exposure increased serum testosterone and decreased sperm motility, but no treatment-related testis histopathology was observed. We hypothesized that gene expression profiles would identify potential mechanisms of toxicity and used DNA microarrays and quantitative real-time PCR (qPCR) to generate profiles. Triazole fungicides are designed to inhibit fungal cytochrome P450 (CYP) 51 enzyme but can also modulate the expression and function of mammalian CYP genes and enzymes. Triazoles affected the expression of numerous CYP genes in rat liver and testis, including multiple Cyp2c and Cyp3a isoforms as well as other xenobiotic metabolizing enzyme (XME) and transporter genes. For some genes, such as Ces2 and Udpgtr2, all four triazoles had similar effects on expression, suggesting possible common mechanisms of action. Many of these CYP, XME and transporter genes are regulated by xeno-sensing nuclear receptors, and hierarchical clustering of CAR/PXR-regulated genes demonstrated the similarities of toxicogenomic responses in liver between all four triazoles and in testis between myclobutanil and triadimefon. Triazoles also affected expression of multiple genes involved in steroid hormone metabolism in the two tissues. Thus, gene expression profiles helped identify possible toxicological mechanisms of the triazole fungicides.

BACKGROUND: Semen quality is associated with fertility status, but there is little quantitative information on risk factors that affect semen quality, especially in non-clinical populations. Advancing male age has been associated with a decline in semen quality, with the largest effect being on sperm motility. However, there is little quantitative data on the specific components of sperm motion that are affected by male age. METHODS: We performed linear regression analyses of 14 aspects of semen quality measured by computer-assisted semen analysis (CASA) in a non-clinical cohort of 90 non-smoking men, aged 22–80 years, who had no history of infertility or reproductive problems. RESULTS: We found age-associated declines in CASA-determined motility (% motile, 0.8% per year; % progressively motile, 0.9% per year; % rapidly motile, 0.4% per year, P £ 0.001) and three quantitative aspects of sperm motion [linearity (LIN), 0.2% per year; straight line velocity (VSL), 0.2% per year, and average path velocity (VAP), 0.3% per year, P < 0.05], with no evidence for age thresholds and no significant association with abstinence duration. Age was not significantly associated with amplitude of lateral head (ALH) displacement, beat cross frequency (BCF) and nuclear elongation or size. CONCLUSIONS: Quantitative analysis of sperm motion indicates that as men age, they produce fewer motile sperm, which are able to travel less along a linear path, thus covering less forward distance per unit time. These findings may have fertility implications for men who choose to delay fatherhood.

A byproduct of drinking water disinfection, bromochloroacetic acid (BCA), acts as a reproductive toxicant in rats. To determine if BCA produces similar reproductive toxicity in mice, juvenile and adult C57BL/6 males were exposed to 0, 8, 24, 72 or 216 mg/kg of BCA once daily for 14 days. Five of 12 animals from each dose-group were sacrificed at the end of dosing, and testes, epididymes, and seminal vesicles harvested and weighed. Seven mice from each dose-group (including juvenile-exposed mice, following a 14-week maturation period) were used in a 40-day sequential breeding assay to determine if BCA targets a particular phase of spermatogenesis. No significant effects were observed in mice exposed to BCA as juveniles, and there were no effects on fertility by 14 weeks after dosing. However, effects were observed in adult-exposed mice over the first 10 days after BCA exposure: mean number of litters/male, percentage of litters/female bred, and total number of fetuses/male were all reduced by 72 and 216 mg/kg BCA. These results in adult mice indicate BCA disrupted differentiation of spermatids during dosing and the first 10 days of mating, and are consistent with the spermatid retention and atypical residual bodies observed in animals exposed to 72 and 216 mg/kg BCA. To investigate mechanisms involved, we utilized cDNA microarrays containing 950 testis-expressed genes to profile gene expression from Control and BCA-treated mice. Statistical analyses of microarray results identified 40 well-characterized genes differentially expressed in a dose responsive manner as a result of BCA exposure. Microarray results were supplemented with quantitative real-time PCR and Westerns for several genes and proteins. The 40 genes whose expression was altered by BCA are involved in numerous biological processes including: cell communication and adhesion, cell cycle and cell proliferation, metabolism, signal transduction, stress response, and spermatogenesis and male fertility. Modulated expression of these genes, particularly the 15 expressed in Sertoli cells and spermatids, offers new insights into potential mechanisms of BCA toxicity in the mouse testis.

The father, like the mother, can transmit genetic defects to his offspring that are detrimental for normal development and a healthy life. Epidemiological studies have identified associations between several paternal exposures and abnormal reproductive outcomes, but these types of studies are inherently complex and expensive, and the risk factors for the paternal contribution to abnormal reproductive outcomes remain poorly understood. Several sensitive methods have been developed for detecting mutations and chromosomal damage directly in sperm. These assays are potential bioindicators for paternal risk factors for infertility, spontaneous abortions, aneuploidy syndromes, and genetic diseases in children. Among these methods, fluorescence in situ hybridization (FISH) has been adapted for the detection of numerical and structural chromosomal abnormalities in the sperm of an expanding number of species, including humans and rodents. Sperm FISH has identified several potential paternal risk factors such as age, drugs, lifestyles, and various environmental/occupational exposures. Here, we summarize the status of the development and usage of these sperm-FISH assays and suggest strategies for prioritizing chemical agents for epidemiological investigations to assess paternal risk for abnormal reproductive outcome.

The chlorination of drinking water results in production of numerous disinfection by-products (DBPs). One of the important classes of DBPs is the haloacetic acids. We have previously shown that the haloacetic acids (HAs), dichloro (DCA), dibromo (DBA) and bromochloro (BCA) acetic acid are developmentally toxic in mouse whole embryo culture. Human exposure to these contaminants in drinking water would involve simultaneous exposure to all three HAs. This study explores the question of developmental toxicity interactions between these compounds. Gestational day (GD) 9.5 rat embryos were exposed to various concentrations of the three HAs (singly or in combination) for 48 h and then evaluated for dysmorphology. The embryonic effects from exposure to the single compounds and mixtures were evaluated using developmental score (DEVSC) as the parameter of comparison. Concentrations of individual compounds and mixtures were chosen (based on a dose-additivity model) which were predicted to produce scores 10 or 20% lower than control levels. Evaluations were performed on all possible combinations of the three HAs. The HAs were dysmorphogenic and resulted in primarily rotation and heart defects and to a lesser extent prosencephalic, visceral arch, and eye defects. The percent anomalies in the rat were comparable to those previously published for the mouse at comparable toxicant concentration. There was a low incidence of neural tube defects in the rat following exposure to the HAs. The rat neural tube appeared less sensitive to the HAs than did the mouse resulting in a higher rate of neural tube dysmorphology in the mouse. Following exposures to BCA and DBA, alone and in combination, there was a significant incidence of delayed embryonic caudal development with apparent normal development anterior to the second visceral arch. The developmental scores for embryos exposed to combinations of the three compounds, when compared to scores for embryos exposed to the single compounds, indicated that the dose-additivity model adequately predicted the observed toxicity and that the developmental toxicity of these water disinfection by-products appears to be additive in whole embryo culture (WEC).

In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm significantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 micro M DX. At 1.6 micro M the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a significant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 micro M DX, continuing at the same level to a final concentration of 1.6 micro M. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 micro M DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the findings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the first reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different end-points, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a 'parallelogram' approach in human cells.

BACKGROUND: Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of ICSI, which can enable men with severely impaired sperm production to father children. Several papers have been published reporting aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients. METHODS: We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolour ACM fluorescence in situ hybridization (FISH) assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen-1q12 region. RESULTS: There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen-1q12 region. There was no evidence for an increase in chromosome 1 disomy, or in diploidy. CONCLUSIONS: Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities, suggesting that oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.

In utero, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure causes abnormal ventral, dorsolateral, and anterior prostate development in C57BL/6J mice. Androgens, mesenchymal-epithelial interactions, and growth factor expression all have roles in initiating and regulating development and growth of the prostate. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), both of which bind the EGF receptor (EGFR), are expressed in human and rodent developing prostate. This study examines the influence of null expression of EGF and/or TGF-alpha on prostatic bud development and on the ability of TCDD to inhibit prostatic budding. Growth factor knockout (-/-) and wild-type (WT) mice were exposed either to vehicle or to TCDD (0, 0.2, 1, 5, 10, 50, 100, or 150 microg/kg) on gestation day (GD) 12. The number of anterior, dorsal, and lateral prostatic buds (ADLB) and ventral buds (VB) were counted on GD 17.5. Control WT and EGF (-/-) fetuses had similar numbers of ADLB and VB. In control TGF-alpha (-/-) fetuses, the number of ADLBs was higher relative to the C57BL/6J. Control EGF + TGF-alpha (-/-) had poor bud outgrowth, especially in the ADL region. TCDD induced a dose-related decrease in bud formation in all strains with the formation of VBs being more sensitive than ADLBs. The severity of the response depended on growth factor expression, with the most severe effects on VBs in the EGF (-/-) and on ADLBs in the EGF + TGF-alpha (-/-) fetuses. TGF-alpha (-/-) and C57BL/6J fetuses responded to TCDD similarly. In conclusion, EGF and TGF-alpha expression are important for the formation of ADLBs and VBs, and expression of EGF and TGF-alpha affects the ability of TCDD to inhibit prostatic bud formation in a region-specific manner.

Cholesterol is necessary for the proper growth and development of the fetus. Consequently, disruptions in cholesterol biosynthesis lead to abnormal fetal development. It has been shown that in cells exposed to polyunsaturated fatty acids (PUFA), the expressions of genes and activities of enzymes involved in cholesterol synthesis are reduced. Similarly, we found that adult male hamsters fed PUFA-enriched diets had an ≈60% reduction in in vivo hepatic sterol synthesis rates. If fetal tissues respond to PUFA in the same manner as do adult livers, then maternal dietary PUFA could lead to a reduction in fetal sterol synthesis rates and possibly abnormal development. To investigate the impact of maternal dietary fatty acids on fetal sterol synthesis rates, female hamsters were fed diets enriched in various fatty acids before and throughout gestation. In vivo sterol synthesis rates were measured in fetuses at mid- and late gestation. At both gestational stages, dietary PUFA had no effect on fetal sterol synthesis rates. This lack of effect was not a consequence of a lack of PUFA enrichment in fetal fatty acids or the lack of PUFA receptor expression in the fetus. We hypothesize that the fetus may experience a dysregulation of sterol synthesis as the result of the fetus being in a negative sterol balance; the PUFA-induced suppression of sterol synthesis in the adult male hamster liver was ablated by creating a net negative sterol balance across the adult hepatocyte.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate (CP) and hydronephrosis (HN) in mice. The etiology of these defects involves hyperproliferation of epithelial cells of the secondary palatal shelf and ureter, respectively. These effects correlate with altered expression of the epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). In this study, the developmental toxicity of TCDD was examined in EGF, TGF-alpha, and double EGF + TGF-alpha knockout (-/-) and wild type (WT) mice. The influence of background genetics in responsiveness to TCDD was examined using liver 7-ethoxyresorufin-O-deethylase (EROD) activity. Animals were dosed by gavage with 0, 0.2, 1, 5, 24, 50, 100, or 150 micro g TCDD/kg (5 ml/kg) body weight on gestation day 12. The mixed genetic background of WT, EGF (-/-), and EGF + TGF-alpha (-/-) made these mice less responsive to TCDD relative to C57BL/6J and TGF-alpha (-/-), which have a C57BL background. These results show that EGF and TGF-alpha are not required for response to TCDD; however, the specific ligand available to bind EGFR affects the responsiveness to TCDD. EGF (-/-) mice are less responsive for CP, but more sensitive to HN. TGF-alpha (-/-) mice were similar to WT in sensitivity for induction of CP and HN. The responses of EGF + TGF-alpha (-/-) mice were like the WT except at higher doses where sensitivity to CP increased, suggesting that the responses may be mediated by alternative ligands for EGFR that are not functional equivalents of EGF or TGF-alpha. In conclusion, the EGFR pathway is mechanistically important in responses of the embryo to TCDD. Specific ligands confer sensitivity or resistance that are target tissue-dependent.

We have isolated and functionally characterized an additional murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane transport proteins from mouse liver. The 3.6 kb cDNA insert contains an open reading frame of 2010 bp coding for a 670 amino acid protein. Based on its amino acid identity of 88% to the rat Oatp2, it is considered the mouse Oatp2 orthologue. Functional expression in Xenopus laevis oocytes demonstrated that mouse Oatp2 transports several general Oatp substrates such as estrone-3-sulfate, dehydroepiandrosterone sulfate (DHEAS), ouabain and BQ-123 but hardly any taurocholate nor rocuronium or deltorphin II. The high-affinity rat Oatp2 substrate digoxin is transported with a rather low affinity with an apparent Km value of 5.7 μM. Bromosulfophthalein (BSP), a substrate not transported by the rat Oatp2, is transported very well by mouse Oatp2. Northern blot analysis demonstrated a predominant expression in the liver with additional signals in kidney and brain. Using fluorescence in situ hybridization, the Oatp2 gene (gene symbol Slc21a5) was mapped to chromosome 6G1–G3.

To perform an interlaboratory comparison of FISH chromosome painting and to study the time-course of translocations and dicentrics in three accident victims exposed to radiation. Also, to use the data in the validation of the FISH technique as a retrospective dosimeter.Twelve blood samples were collected during 4 years from three subjects exposed to radiation in an accident in Estonia in 1994 involving gamma-radiation from a 137Cs source. Two of the subjects were exposed during approximately 7 h, both receiving a protracted dose of about 1 Gy and also localized exposure. The third subject received a protracted whole-body dose of 2.7 Gy during 4 weeks as well as a short-term partial-body dose. Preparations from 48-h metaphase cultures were painted by the FISH technique using routine methods and probe cocktails in four laboratories. Samples from each subject were analysed in two different laboratories that used different combinations of whole chromosome probes. The PAINT nomenclature was applied when recording chromosome aberrations.The intercomparison of FISH analysis data showed reasonable similarities between laboratories, the largest discrepancy being 21% in the frequency of two-way translocations in subject 3. Half-time calculations, based on combined data sets from two laboratories, showed that dicentrics decreased rapidly with half-times of approximately 2 years. In all cases, the initial dicentric yields were lower than the initial translocation yields. During the 4-year follow-up, the frequencies of all translocations in cells containing only simple rearrangements fell on average to about 65% of their initial value. Two-way translocations were slightly more persistent than all translocations. The average half-time was about 8 years for two-way translocations and around 6 years for all translocations. Cells containing complex rearrangements were few in number and they disappeared with time. In general, the inclusion of complex cells caused a more rapid fall in aberration yield.In general, the results imply that relatively consistent scoring data were obtained with different chromosome painting protocols. They also support the idea that the reduction of translocations with time is associated with partial-body irradiation.

Sudden cardiac death (SCD) in young athletes during physical stress is a rare event with an incidence of 1-3 deaths per 100,000 athletes per year. A coronary anomaly is the second most common cause of death following hypertrophic cardiomyopathy. Symptomatic prodromes occur in 20% of cases prior to the SCD event. This case report describes a 35-year-old male who collapsed near the finishing line of a half marathon run. Despite immediate resuscitation attempts and initial return of spontaneous circulation (ROSC), a pulseless electrical activity (PEA) followed and the patient died 1 h after arrival in the resuscitation unit. The autopsy revealed an anomalous left coronary artery (ALCA), which can lead to ischemia of the respective heart muscles under severe stress.

The aim of the study was to examine risks, implications and outcomes of coronary sinus (CS) lead extraction in patients with infections of cardiac resynchronization therapy (CRT) systems.The study included 40 (65.5 ± 11.1 years; 80% male) transvenous CS lead extraction procedures performed between 2000-2011. Nine (22.5%) patients suffered from infection and included one sepsis (11.1%), two (22.2%) of lead and valve endocarditis, and four (44.4%) cases of pocket infection. CS lead extraction in the infection subgroup was performed between 14 days and more than five years after the last CIED-related surgical procedure.Totally 42 CS and 35 non-CS leads were extracted. Leads extracted in the infection subgroup were significantly longer in situ (49.7 ± 30.7 months) compared to the non-infection subgroup (19.2 ± 28.6 months). Extraction in infected patients required more aggressive methods and longer exposure to radiation than non-infected. Procedural success without major complications was achieved in all patients. Minor post-procedural complications occurred in four (44.4%) of the infected and one (3.2%) of the non-infected patients and were surgical-related in three cases. Overall hospitalization times were significantly longer for the infection than for the non-infection subgroup (21.4 ± 15 versus 9.6 ± 6.9 days).Our results support the concept of complete CIED-system removal in CIED-associated infection, regardless of whether or not infection appears to be limited to the generator pocket site, despite risk of heart failure, patient frailty and a high level of comorbidity. An interdisciplinary approach encompassing appropriate diagnostic, procedural and safety standards allows CS lead extraction in this high-risk subpopulation to be performed with excellent outcomes and low complication rates.

Male-mediated developmental toxicity has been of concern for many years. The public became aware of male-mediated developmental toxicity in the early 1990s when it was reported that men working at Sellafield might be causing leukemia in their children. Human and animal studies have contributed to our current understanding of male-mediated effects. Animal studies in the 1980s and 1990s suggested that genetic damage after radiation and chemical exposure might be transmitted to offspring. With the increasing understanding that there is histone retention and modification, protamine incorporation into the chromatin and DNA methylation in mature sperm and that spermatozoal RNA transcripts can play important roles in the epigenetic state of sperm, heritable studies began to be viewed differently. Recent reports using molecular approaches have demonstrated that DNA damage can be transmitted to babies from smoking fathers, and expanded simple tandem repeats minisatellite mutations were found in the germline of fathers who were exposed to radiation from the Chernobyl nuclear power plant disaster. In epidemiological studies, it is possible to clarify whether damage is transmitted to the sons after exposure of the fathers. Paternally transmitted damage to the offspring is now recognized as a complex issue with genetic as well as epigenetic components.

Aneuploidy studies in sperm such as the sperm-FISH assay require a precise knowledge of the duration of spermatogenesis, especially of the meiotic stages. This is important in order to sample sperm from the epididymis at appropriate intervals after animal treatment. However, aneugens may delay the cell cycle. The progression from meiotic divisions to epididymal sperm was determined by labelling the last S-phase before meiosis with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. In a time frame of 20--24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). We studied the effects of the chemicals acrylamide, colchicine, diazepam, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine on the duration of meiotic divisions in male mice. Colchicine treatment prolonged the duration of meiotic divisions by about 48 h. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine group were 11.7 and 9.4%, respectively, while they were 28.4 and 30.6%, respectively, in the concurrent controls (P > 0.01). On day 24 after treatment, the frequency of labelled sperm in the colchicine group reached the control level. Etoposide treatment resulted in an elevation of BrdU-labelled sperm at 23 rather than 22 days. The other chemicals showed no significant effect of prolonging meiotic cell cycle progression. On the basis of the colchicine and etoposide data, it is suggested that the effect of a chemical on the meiotic cell cycle progression is determined first in order to chose the appropriate sperm sampling time to detect aneuploidy induction.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure produces hydronephrosis and cleft palate in mice. These responses are corre- lated with disruption of expression of epidermal growth factor (EGF) receptor ligands, primarily EGF and transforming growth factor- alpha (TGF-a), and altered epithelial cell proliferation and differen- tiation. This research examined the role of these growth factors in TCDD-induced teratogenicity by using wild type (WT) and knockout (-/-) mice that do not express EGF, TGF-a, or both EGF and TGF-a. Pregnant females were weighed on GD 12 and dosed by gavage with either corn oil or TCDD at 24 mg/kg, 5 ml/kg. On GD 17.5, the maternal parameters evaluated included body weight, body weight gain, liver weight (absolute and adjusted for body weight). The number of implantations, live and dead fetuses, early or late resorp- tions, the proportion of males, fetal body weight, fetal absolute and relative liver weight, placenta weight, incidence of cleft palate, and the severity and incidence of hydronephrosis were recorded. TCDD did not affect maternal weight gain, fetal weight, or survival, but maternal and fetal liver weights and liver-to-body weight ratios were increased in all genotypes. The WT and TGF-a (-/-), but not the EGF (-/-) and EGF 1 TGF-a (-/-) fetuses, developed cleft palate after exposure to 24 mg TCDD/kg. Hydronephrosis was induced by TCDD in all genotypes, with the incidence in EGF 1 TGF-a (-/-) fetuses comparable to that of the WT. The incidence and severity of this defect was substantially increased in EGF (-/-) and TGF-a (-/-). In conclusion, this study demonstrated that expression of EGF influ- ences the induction of cleft palate by TCDD. Also, EGF and TGF-a The environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, dioxin) is the most potent and extensively studied member of the polychlorinated aromatic hydrocarbon (PAH) family of compounds. Members of this family of com- pounds produce similar patterns of toxicity and appear to have a common mechanism of action (Birnbaum et al., 1985; Mc- Connell and Moore, 1979; Poland and Knutson, 1982). TCDD is produced during industrial and combustion processes such as chemical pulp bleaching and waste incineration (Abbott et al., 1987; Firestone et al., 1972). The widespread distribution and stability of TCDD have resulted in bioaccumulation of the compound throughout the environment and food chain (Mc- Connell and Moore, 1979). TCDD has been shown to induce a variety of toxic responses in laboratory animals such as carci- nogenicity, hepatotoxicity, fetotoxicity, reproductive toxicity, and teratogenicity (McConnell et al., 1978; Poland and Knut- son, 1982; Schwetz et al., 1973). Human exposure to TCDD currently occurs primarily via dietary consumption, although there have been several accidental and occupational exposures (Fingerhut et al., 1991; Mastroiacovo et al., 1988; Reggiani, 1989). Because of its relative resistance to metabolism, TCDD tends to persist in the body and its half life in humans is estimated at 7 to 10 years (Pirkle et al., 1989). TCDD is a reproductive and developmental toxicant in wild- life and laboratory animals, including rats, hamsters, and mice. The primary teratogenic abnormalities produced in mice are hydronephrosis and cleft palate (Moore, 1973). In the C57BL/6N mouse, these responses are seen at doses that do not result in maternal or fetal mortality (Birnbaum et al., 1985).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1x10(-8), 1x10(-9) or 1x10(-10) M TCDD. Exposure to 1x10(-10) M TCDD did not affect the cultures, while 1x10(-8) and 1x10(-9) M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.

Aneuploidy induction in male germ cells of mice and men after chronic exposure to diazepam (DZ; CAS 439-14-5; Valium was assessed by multicolor fluorescence in situ hybridization (FISH). DZ, a widely administered sedative and muscle relaxant, was proposed to act as an aneugen by disturbing spindle function in various assay systems. Male mice were treated by oral intubation with 3mg/kg DZ once or daily for 14 consecutive days. At 22 days after the last treatment, epididymal sperm were collected from the caudae epididymes. Evaluation of aneuploid and diploid sperm (10,000 sperm per animal) was performed by multicolor FISH employing DNA probes specific for chromosomes X, Y, and 8 simultaneously. We found a significant increase in the frequency of disomy 8 in subchronically DZ-treated mice when compared to the concurrent solvent control group (2.4-fold; P0.01), while no increase was detected for sex-chromosome hyperhaploidies. No effect was seen when mice were treated with a single dose (3mg/kg DZ). In a parallel human approach, two men were evaluated who chronically ingested0.3mg/kg/d DZ for more than 6 months. Multicolor FISH was applied to human sperm probing for chromosomes X, Y, and 13. Frequencies for sperm with disomy 13, disomy X, and total sex-chromosomal disomies were found to be elevated among the two subjects after chronic DZ-exposure compared to control subjects. In conclusion, the results indicate that diazepam acts as an aneugen during meiosis in male spermatogenesis, both in mice and humans. The quantitative comparison indicates that humans may be at least 10 times more sensitive than mice for aneuploidy induction by DZ during male meiosis.

In order to assess the effects of trichlorfon on cell division and on aneuploidy induction, we conducted an in vitro assay for spindle disturbances using V79 cells and an in vivo assay for aneuploidy induction in meiosis of male mice using multicolour fluorescence in situ hybridization (FISH) with epididymal sperm. In the in vitro assay, the chemical caused a concentration-dependent increase in the incidence of initial and full c-mitoses in the dose range 40-120 microg/ml trichlorfon. The mitotic index (MI) was decreased between 40 and 100 microg/ml trichlorfon, whereas at 120 microg/ml the MI was back to the control level, coinciding with the dramatic increase in c-mitoses. The results confirm that trichlorfon is a potent spindle poison in V79 cells. In the in vivo multicolour FISH assay, administration of trichlorfon to male mice at single doses of 200, 300 and 405 mg/kg caused a dose-dependent increase of the frequencies of disomic sperm (0.068, 0.074 and 0.134%, respectively) compared with the corresponding controls (0.046, 0.042 and 0.056%, respectively). The prevalence of X-X-8 and Y-Y-8 sperm suggests that trichlorfon affected chromosome segregation predominantly during the second meiotic division. Diploid sperm were not induced by trichlorfon treatment, indicating that no meiotic block occurred. It is concluded that trichlorfon is a potent spindle poison in V79 cells and induces aneuploidy in mouse spermatocytes during meiosis.

Capsular block syndrome (CBS) is a complication of uncomplicated cataract surgery. Depending on etiology and the time of onset CBS can be subdivide into three types. The cases of 4 patients with CBS type III who presented with a milky fluid in the space between the posterior surface of the implanted intraocular lens (IOL) and the anterior surface of the distended posterior capsular bag are described. All patients complained of reduced vision. The therapy of choice is Nd:YAG laser posterior capsulotomy but with suspected low-grade endophthalmitis surgical intervention can also be carried out and submitting the plug for bacterial culture.

Multicolor fluorescence in situ hybridization (FISH) was used to investigate the induction of aneuploidy during meiosis in young adult male mice treated with chemicals chosen for the EU sponsored aneuploidy project (acrylamide, colchicine, diazepam and thiabendazole). The aim of the present study was to evaluate the frequency of aneuploid sperm induced by each of these chemicals by sperm FISH. Male (102/ElxC3H/El)F1 mice were treated with acrylamide (120 and 60 mg/kg single dose i.p.), colchicine (1.5 and 3 mg/kg single dose, i.p.), diazepam (300, 150 and 75 mg/kg single dose by oral intubation) or thiabendazole (100 and 300 mg/kg daily for 11 days by oral intubation). At 22 days after the last treatment, sperm were collected from the cauda epididymis. Three chromosome FISH was applied to determine hyperhaploid and diploid sperm with DNA probes specific for the chromosomes X, Y and 8. Five animals were treated per dose group and sperm aneuploidy was evaluated in 10,000 sperm per animal. We found significant increases in the frequency of total hyperhaploidy for the males treated with 3.0 mg/kg colchicine (0.092 versus 0.056%, P < 0.05) and with 1.5 mg/kg colchicine (0.082 versus 0.050%, P < 0.05), as well for the males treated with 300 mg/kg diazepam (0.081 versus 0.050%, P < 0.05), indicating that colchicine and diazepam each induced germ cell aneuploidy. We also found significant increases in the frequency of total diploidy for the males treated with 300 mg/kg diazepam (P < 0.05) and with 300 mg/kg thiabendazole (P < 0.05). No significant effects were found for 120 and 60 mg/kg acrylamide or for the other doses of diazepam and thiabendazole. These first results indicate that the multicolor FISH method is useful to determine aneuploidy induction in sperm of mice.

The aryl hydrocarbon receptor (AHR) is a transcriptional regulatory protein that binds to upstream DNA response elements of target genes. Activation of the AHR by binding of ligands such as polyhalogenated dioxins, furans, and PCBs is associated with a wide range of adverse biological outcomes, including cancer, immune deficiencies, embryo/fetotoxicity, and reproductive toxicity. Investigations of the diverse biological responses mediated by the AHR led to production of a transgenic mouse in which the gene coding for the AhR was inactivated. AHR-deficient mice were fertile and at maturity exhibited immune system impairment and hepatic fibrosis. Our laboratory received several of these homozygous knockout (−/−) mice and mated them with wild-type (+/+) C57BL/6N mice to generate large numbers of heterozygotes (+/−). The −/− males were then mated with a total of 45 heterozygous +/− females. Offspring of these matings were genotyped and mated in all genotypic combinations. Although male and female −/− adults were fertile, the −/− females had difficulty maintaining conceptuses during pregnancy, surviving pregnancy and lactation, and rearing pups to weaning. Only 46% of the 39 pregnant −/− females successfully raised pups to weaning. The −/− pups showed poor survival during lactation (average death rate per litter was 16%) and after weaning (26.5% of the 230 weaned −/− pups died within 2 weeks). Only 39% of the implantations in uteri of −/− dams resulted in offspring surviving to Postnatal Day 45. Across all litters the sex ratios and genotypic frequencies were comparable to expected values. Reproductive success was adversely affected inAhr-null females and conceptuses. Additional study is needed to reveal the etiology of these effects.