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Reasons for Performing Study: Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known., Objectives: The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies., Study Design: In vivo experiment., Methods: Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n = 8) and immunoblotting for protein expression (n = 3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7 days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05., Results: Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT., Conclusions: Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation., (© 2015 EVJ Ltd.)

This workshop reviewed the current science to inform and recommend the best evidence-based approaches on the use of germ cell genotoxicity tests. The workshop questions and key outcomes were as follows. (1) Do genotoxicity and mutagenicity assays in somatic cells predict germ cell effects? Limited data suggest that somatic cell tests detect most germ cell mutagens, but there are strong concerns that dictate caution in drawing conclusions. (2) Should germ cell tests be done, and when? If there is evidence that a chemical or its metabolite(s) will not reach target germ cells or gonadal tissue, it is not necessary to conduct germ cell tests, notwithstanding somatic outcomes. However, it was recommended that negative somatic cell mutagens with clear evidence for gonadal exposure and evidence of toxicity in germ cells could be considered for germ cell mutagenicity testing. For somatic mutagens that are known to reach the gonadal compartments and expose germ cells, the chemical could be assumed to be a germ cell mutagen without further testing. Nevertheless, germ cell mutagenicity testing would be needed for quantitative risk assessment. (3) What new assays should be implemented and how? There is an immediate need for research on the application of whole genome sequencing in heritable mutation analysis in humans and animals, and integration of germ cell assays with somatic cell genotoxicity tests. Focus should be on environmental exposures that can cause de novo mutations, particularly newly recognized types of genomic changes. Mutational events, which may occur by exposure of germ cells during embryonic development, should also be investigated. Finally, where there are indications of germ cell toxicity in repeat dose or reproductive toxicology tests, consideration should be given to leveraging those studies to inform of possible germ cell genotoxicity., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Background: Stunting is prevalent by the age of 6 months in the indigenous population of the Western Highlands of Guatemala., Aim: The objective of this study was to determine the time course and predictors of linear growth failure and weight-for-age in early infancy., Study Design and Subjects: One hundred and forty eight term newborns had measurements of length and weight in their homes, repeated at 3 and 6 months. Maternal measurements were also obtained., Results: Mean ± SD length-for-age Z-score (LAZ) declined from newborn -1.0 ± 1.01 to -2.20 ± 1.05 and -2.26 ± 1.01 at 3 and 6 months respectively. Stunting rates for newborn, 3 and 6 months were 47%, 53% and 56% respectively. A multiple regression model (R(2) = 0.64) demonstrated that the major predictor of LAZ at 3 months was newborn LAZ with the other predictors being newborn weight-for-age Z-score (WAZ), gender and maternal education∗maternal age interaction. Because WAZ remained essentially constant and LAZ declined during the same period, weight-for-length Z-score (WLZ) increased from -0.44 to +1.28 from birth to 3 months. The more severe the linear growth failure, the greater WAZ was in proportion to the LAZ., Conclusion: The primary conclusion is that impaired fetal linear growth is the major predictor of early infant linear growth failure indicating that prevention needs to start with maternal interventions., (© 2013.)

Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent-offspring trios from highly exposed human populations, and controlled dose-response experiments in animals. These studies will establish background levels of variability in germline mutation rates and identify environmental agents that influence these rates and heritable disease. Guidance for the types of exposures to examine come from rodent studies that have identified agents such as cancer chemotherapeutic drugs, ionizing radiation, cigarette smoke, and air pollution as germ-cell mutagens. Research is urgently needed to establish the health consequences of parental exposures on subsequent generations., (Published by Elsevier B.V.)

Deriving No Observed Adverse Effect Level (NOAEL) or benchmark dose is important for risk assessment and can be influenced by study design considerations. In order to define the di-(2-ethylhexyl) phthalate (DEHP) dose-response curve for reproductive malformations, we retained more offspring to adulthood to improve detection of these malformations in the reproductive assessment by continuous breeding study design. Sprague-Dawley rats were given a dietary administration of 1.5 (control), 10, 30, 100, 300, 1000, 7500, and 10,000 ppm DEHP. Male pups were evaluated for gross reproductive tract malformations (RTMs) associated with the "phthalate syndrome." DEHP treatment had minimal effects on P0 males. There was a statistically significant increase in F1 and F2 total RTMs (testis, epididymides, seminal vesicle, and prostate) in the 7500-ppm dose group and F1 10,000-ppm dose group. The 10,000-ppm exposed F1 males did not produce an F2 generation. The NOAEL for F1 and F2 RTM combined data, because in utero exposures were similar, were 100 ppm (4.8 mg/kg/day), which was close to the 5% response benchmark dose lower confidence limit of 142 ppm. The utility of evaluating more pups per litter was examined by generating power curves from a Monte Carlo simulation. These curves indicate a substantial increase in detection rate when three males are evaluated per litter rather than one. A 10% effect across male pups would be detected 5% of the time if one pup per litter was evaluated, but these effects would be detected 66% of the time if three pups per litter were evaluated. Taken together, this study provides a well-defined dose response of DEHP-induced RTMs and demonstrates that retention of more adult F1 and F2 males per litter, animals that were already produced, increases the ability to detect RTMs and presumably other low-incidence phenomena.

Background: Common psychological adjustment difficulties have been identified for groups of implantable cardioverter defibrillator patients, such as those who are young (<50 years old), have been shocked, and are female. Specific aspects and concerns, such as fears of death or shock and body image concerns, that increase the chance of distress, have not been examined in different aged female implantable cardioverter defibrillator (ICD) recipients. The aim of the study was to investigate these areas of adjustment across three age groups of women from multiple centers., Methods: Eighty-eight female ICD patients were recruited at three medical centers: Shands Hospital at the University of Florida, Brigham and Women's Hospital in Boston, and Royal North Shore Hospital in Sydney, Australia. Women completed individual psychological assessment batteries, measuring the constructs of shock anxiety, death anxiety, and body image concerns. Medical record review was conducted for all patients regarding cardiac illnesses and ICD-specific data., Results: Multivariate and univariate analyses of variance revealed that younger women reported significantly higher rates of shock and death anxiety (Pillai's F=3.053, P=0.018, eta2p=0.067) and significantly greater body image concerns (Pillai's F=4.198, P=0.018, eta2p=0.090) than middle- and older-aged women., Conclusions: Women under the age of 50 appear to be at greater risk for the development of psychosocial distress associated with shock anxiety, death anxiety, and body image. Clinical-based strategies and interventions targeting these types of adjustment difficulties in younger women may allow for improved psychosocial and quality of life outcomes.

Zidovudine-based antiretroviral therapies (ARTs) for treatment of HIV-infected pregnant women have markedly reduced mother-to-child transmission of the human immunodeficiency virus (HIV-1) from approximately 25% to <1%. However, zidovudine (ZDV; AZT), a nucleoside analogue, induces chromosomal damage, gene mutations, and cancer in animals following direct or transplacental exposure. To determine if chromosomal damage is induced by ZDV in infants exposed transplacentally, we evaluated micronucleated reticulocyte frequencies (%MN-RET) in 16 HIV-infected ART-treated mother-infant pairs. Thirteen women received prenatal ART containing ZDV; three received ART without ZDV. All infants received ZDV for 6 weeks postpartum. Venous blood was obtained from women at delivery and from infants at 1-3 days, 4-6 weeks, and 4-6 months of life; cord blood was collected immediately after delivery. Ten cord blood samples (controls) were obtained from infants of HIV-uninfected women who did not receive ART. %MN-RET was measured using a single laser 3-color flow cytometric system. Tenfold increases in %MN-RET were seen in women and infants who received ZDV-containing ART prenatally; no increases were detected in three women and infants who received prenatal ART without ZDV. Specifically, mean %MN-RET in cord blood of ZDV-exposed infants was 1.67 +/- 0.34 compared with 0.16 +/- 0.06 in non-ZDV ART-exposed infants (P = 0.006) and 0.12 +/- 0.02 in control cord bloods (P < 0.0001). %MN-RET in ZDV-exposed newborns decreased over the first 6 months of life to levels comparable to cord blood controls. These results demonstrate that transplacentalZDV exposure is genotoxic in humans. Long-term monitoring of HIV-uninfected ZDV-exposed infants is recommended to ensure their continued health., ((c) 2006 Wiley-Liss, Inc.)

Antiretroviral therapies based on nucleoside reverse transcriptase inhibitors (NRTIs), like zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), markedly reduce mother-to-child transmission of the human immunodeficiency virus (HIV). However, AZT induces damage in nuclear DNA of mice exposed in utero and postnatally, and mitochondrial DNA (mtDNA) damage has been observed in both human and mouse neonates following perinatal exposure to AZT and AZT/3TC in combination. To provide animal data modeling the NRTI-induced heart damage reported in human infants, we treated pregnant CD-1 mice throughout gestation and treated their pups by direct gavage from postnatal day (PND) 4 through PND 28 with daily doses of 150 mg/kg body weight (bw)/day AZT, 75 mg/kg bw/day 3TC, 125/62.5 mg/kg bw/day AZT/3TC, or the vehicle control. Half the pups were euthanized on PND 28; the remainder received no further dosing, and were euthanized at week 10. Heart tissue was collected, total DNA was extracted, and mtDNA copy number relative to nuclear DNA copy number, mtDNA damage, and mtDNA mutation assays were performed using PCR-based methods. Analyses revealed increases in mtDNA lesions in 4-week-old males and females treated with AZT or 3TC, but not in 10-week-old mice, suggesting that the damage resolved after treatment ceased. Interestingly, 10-week-old females treated with AZT/3TC had significant increases in mtDNA damage. Point mutations were elevated in 10-week-old females treated with AZT or AZT/3TC, but not 3TC; no increases in mutations were seen in either gender at 4 weeks of age. Our data suggest that AZT/3TC combination treatment produces greater mtDNA damage than either agent individually, and that female mice are more sensitive than males to AZT/3TC-induced mtDNA damage., ((c) 2006 Wiley-Liss, Inc.)

Background: Etoposide (ET) is a chemotherapeutic agent widely used in the treatment of leukaemia, lymphomas and many solid tumours such as testicular and ovarian cancers, all of which are common in patients of reproductive age. The purpose of the study was to characterize the long-term effects of ET on male germ cells using sperm fluorescence in situ hybridization (FISH) analyses., Methods: Chromosomal aberrations (partial duplications and deletions) and whole chromosomal aneuploidies were detected in sperm of mice treated with a clinical dose of ET. Semen samples were collected at 25 and 49 days after dosing to investigate the effects of ET on meiotic pachytene cells and spermatogonial stem-cells, respectively., Results: ET treatment resulted in major increases in the frequencies of sperm-carrying chromosomal aberrations in both meiotic pachytene (27- to 578-fold) and spermatogonial stem-cells (8- to 16-fold), but aneuploid sperm were induced only after treatment of meiotic cells (27-fold) with no persistent effects in stem cells., Conclusion: These results show that ET may have long-lasting effects on the frequencies of sperm with structural aberrations. This has important implications for cancer patients undergoing chemotherapy with ET because they may remain at higher risk for abnormal reproductive outcomes long after the end of chemotherapy.

We developed a live high-field multiple-mouse magnetic resonance imaging method to increase the throughput of imaging studies involving large numbers of mice. Phantom experiments were performed in 7 shielded radiofrequency (RF) coils for concurrent imaging on a 7 Tesla MRI scanner outfitted with multiple transmit and receive channels to confirm uniform signal-to-noise ratio and minimal ghost artifacts across images from the different RF coils. Grid phantoms were used to measure image distortion in different positions in the coils. The brains of 7 live mice were imaged in 3D in the RF coil array, and a second array of 16 RF coils was used to 3D image the whole bodies of 16 fixed, contrast agent-perfused mice. The images of the 7 live mouse brains at 156 microm isotropic resolution and the 16 whole fixed mice at 100 microm isotropic resolution were of high quality and free of artifacts. We have thus shown that multiple-mouse MRI increases throughput for live and fixed mouse experiments by a factor equaling the number of RF coils in the scanner., ((c) 2005 Wiley-Liss, Inc.)

Acrylamide is an animal carcinogen and probable human carcinogen present in appreciable amounts in heated carbohydrate-rich foodstuffs. It is also a germ cell mutagen, inducing dominant lethal mutations and heritable chromosomal translocations in postmeiotic sperm of treated mice. Acrylamide's affinity for male germ cells has sometimes been overlooked in assessing its toxicity and defining human health risks. Previous investigations of acrylamide's germ cell activity in mice showed stronger effects after repeated administration of low doses compared with a single high dose, suggesting the possible involvement of a stable metabolite. A key oxidative metabolite of acrylamide is the epoxide glycidamide, generated by cytochrome P4502E1 (CYP2E1). To explore the role of CYP2E1 metabolism in the germ cell mutagenicity of acrylamide, CYP2E1-null and wild-type male mice were treated by intraperitoneal injection with 0, 12.5, 25, or 50 mg acrylamide (5 ml saline)(-1) kg(-1) day(-1) for 5 consecutive days. At defined times after exposure, males were mated to untreated B6C3F1 females. Females were killed in late gestation and uterine contents were examined. Dose-related increases in resorption moles (chromosomally aberrant embryos) and decreases in the numbers of pregnant females and the proportion of living fetuses were seen in females mated to acrylamide-treated wild-type mice. No changes in any fertility parameters were seen in females mated to acrylamide-treated CYP2E1-null mice. Our results constitute the first unequivocal demonstration that acrylamide-induced germ cell mutations in male mice require CYP2E1-mediated epoxidation of acrylamide. Thus, CYP2E1 polymorphisms in human populations, resulting in variable enzyme metabolic activities, may produce differential susceptibilities to acrylamide toxicities.

Zidovudine (ZDV), an antiretroviral drug used alone or in combination with other antiretroviral agents to treat HIV-infected pregnant women and their newborn infants, effectively reduces mother-to-child transmission of the virus. That myopathy and cardiomyopathy, related to mitochondrial damage, develop in some adults chronically treated with ZDV has long been known; recently, reports have suggested that similar adverse effects may occur in some infants exposed perinatally. Using a mouse model of human neonatal exposure, we treated pregnant CD-1 mice twice daily with doses of 75 mg/kg ZDV plus 37.5 mg/kg lamivudine throughout gestation and lactation; pups were exposed by direct gavage beginning postnatal day (PND) 4 and sacrificed on PND 28. Hearts were removed rapidly, and ventricles were processed for electron microscopy. Morphometric and semiquantitative morphological analyses were performed on three micrographs from each of three blocks from each of three females and three males from the control and treated groups. Treated mice showed significant increases in the mean area and decreases in the mean number of cardiomyocytic mitochondria compared to controls. We observed clusters of damaged mitochondria more frequently in treated animals than in controls; damage included fragmentation and loss of cristae. These results, demonstrating alterations in cardiomyocytic mitochondria of mice exposed in utero and postnatally, may model cardiac damage reported in human infants similarly exposed to ZDV. Critical insights derived from animal-model data like these may be used to mitigate risks to thousands of human infants receiving essential lifesaving therapy with antiretroviral drugs.

The developmental consequences of chromosomal aberrations in embryos include spontaneous abortions, morphological defects, inborn abnormalities, and genetic/chromosomal diseases. Six germ-cell mutagens with different modes of action and spermatogenic stage sensitivities were used to investigate the relationship between the types of cytogenetic damage in zygotes with their subsequent risk of postimplantation death and of birth as a translocation carrier. Independent of the mutagen used, over 98% of paternally transmitted aberrations were chromosome type, rather than chromatid type, indicating that they were formed during the period between exposure of male germ cells and initiation of the first S phase after fertilization. There were consistent one-to-one agreements between the proportions of a) zygotes with unstable aberrations and the frequencies of dead embryos after implantation (slope = 0.87, confidence interval [CI]: 0.74, 1.16) and b) zygotes with reciprocal translocations and the frequency of translocation carriers at birth (slope = 0.74, CI: 0.48, 2.11). These findings suggest that chromosomal aberrations in zygotes are highly predictive of subsequent abnormal embryonic development and that development appears to proceed to implantation regardless of the presence of chromosomal abnormalities. Our findings support the hypothesis that, for paternally transmitted chromosomal aberrations, the fate of the embryo is already set by the end of G1 of the first cell cycle of development.

Human immunodeficiency virus (HIV)-infected pregnant women are administered nucleoside-analogue antiretrovirals to reduce maternal-infant viral transmission. The current protocol recommends treating newborns for 6 additional weeks postpartum. The treatment is effective, but the risk of drug-induced chromosomal damage in neonates remains undefined. We used a mouse model to investigate this concern. In a multigeneration reproductive toxicity study, female CD-1 mice received 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI) (50/250, 75/375, 150/750 mg/kg/day AZT/ddI) by gavage twice daily in equal fractions beginning prior to mating and continuing throughout gestation and lactation. Direct pup dosing (same regimen) began on postnatal day (PND) 4. Peripheral blood erythrocytes of male pups were screened for micronuclei, markers of chromosomal damage, on PNDs 1, 4, 8, and 21. Extraordinary increases in micronucleated cells were noted in pups for each treatment group at each sampling time; treated dams exhibited smaller yet significant increases in micronucleated erythrocytes. The frequencies of micronucleated cells in untreated pups were higher than in the untreated dams, and all pups had markedly elevated levels of circulating reticulocytes compared to dams. These observations suggest that fetal and neonatal mouse hematopoietic precursor cells have heightened sensitivity to genotoxic agents, perhaps due to rapid cell proliferation during the perinatal period of development. The amount of genetic damage observed in treated pups raises concern for the potential of similar damage in humans. Investigations of chromosomal integrity in exposed newborns and children are recommended., (Copyright 2004 Wiley-Liss, Inc.)

We previously reported extraordinary increases in micronucleated erythrocytes in CD-1 mouse pups exposed to 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI; 50/250, 75/375, 150/750 mg/kg/day AZT/ddI) by gavage throughout gestation and lactation, followed by direct pup dosing beginning postnatal day (PND) 4 (Bishop et al. [2004]: Environ Mol Mutagen 43: 3-9). That study was conducted to explore the potential for genetic damage in newborns exposed perinatally to antiretrovirals in order to reduce maternal-infant transmission of HIV-1. Because dramatic increases in frequencies of micronucleated erythrocytes were seen in exposed pups, additional studies were conducted to clarify the relative contribution of each drug to the observed damage. Pregnant CD-1 mice were administered AZT (50, 75, 150 mg/kg/day) or ddI (250, 375, 750 mg/kg/day) by gavage twice daily in equal fractions beginning prior to mating and continuing throughout gestation and lactation. Direct pup dosing (same regimens) began on PND 4. Peripheral blood erythrocytes of male pups were screened for micronuclei on PNDs 1, 4, 8, and 21. Significant increases in micronucleated erythrocytes were observed in pups and dams exposed to AZT at all doses and sampling times. The highest micronucleus levels were observed in pups on PND 8 after the initiation of direct dosing. In contrast, effects seen in pups and dams treated with ddI were minimal. These results demonstrate that AZT, a component of many anti-HIV combination therapies, induces chromosomal damage in perinatally exposed neonatal mice. Comparison of micronucleated cell frequencies induced by AZT alone or in combination with ddI suggests that ddI potentiates AZT-induced chromosomal damage following direct exposure.

N-Hydroxymethylacrylamide (NHMA), a mouse carcinogen inactive in the Salmonella assay and mouse micronucleus (MN) assay, was tested for reproductive effects in a mouse continuous breeding study. In that study, increased embryonic deaths were observed after 13 weeks exposure of parental animals to NHMA via drinking water (highest dose, 360 ppm); the results indicated the possible induction of chromosome damage in germ cells of treated males. An additional mouse MN test was conducted using a 31-day treatment period to better match the dosing regimen used in the breeding study; the results were negative. Additional studies were conducted to explore the germ cell activity of NHMA. A male mouse dominant lethal study was conducted using a single intraperitoneal injection of 150 mg/kg NHMA; the results were negative. A follow-up study was conducted using fractionated dosing, 50 mg/kg/day for 5 days; again, no increase in dominant lethal mutations was observed. NHMA (180-720 ppm) was then administered to male mice in drinking water for 13 weeks, during which three sets of matings occurred. Two weeks after mating, females were killed and the uterine contents were analyzed. Large, dose-related increases in dominant lethal mutations were observed with increasing length of exposure. The magnitude of the increases stabilized after 8 weeks of treatment. However, the frequency of micronucleated peripheral blood erythrocytes was not elevated in mice treated for 13 weeks with NHMA in drinking water. Thus, NHMA appears to be unique in inducing genetic damage in germ cells but not somatic cells of male mice.

Females do differ from males in their germ cell and general reproductive responses to toxicants. Chromatin structure is perhaps one factor that contributes to sex differences in germ cell response to toxicants. Differences in available targets likely contribute to response differences between sexes as well as to within sex differences between germ cell stages. It is important to consider these differences when conducting reproductive toxicity studies and interpreting the results.

The objective of this research was to determine whether Novantrone, Oncovin, Velban, and Prednisone (NOVP) combination chemotherapy for Hodgkin's disease increases the frequencies of the specific types of aneuploid sperm that might elevate the risk of fathering a child with one of the major clinical aneuploidy syndromes, i.e., Down (disomy 21 sperm), Edward (disomy 18 sperm), Turner (nullisomy sex sperm), XYY (disomy Y sperm), triple X (disomy X sperm), or Klinefelter (XY sperm). A four-chromosome multicolor sperm fluorescence in-situ hybridization assay that simultaneously evaluates chromosomes 18, 21, X, and Y was applied to semen provided by four healthy men and to repeated samples of eight Hodgkin's disease patients before treatment, 35-50 days after treatment to examine the effects of treatment on male meiotic cells, and 1-2 years after treatment to measure the persistence of damage. There were chromosome-specific variations in baseline frequencies and significant inductions of all of the detectable types of sperm aneuploidies: XY sperm (14-fold increase), disomy 18 (7-fold), nullisomy sex (3-fold), disomy 21 (3-fold), and disomy X and Y (approximately 2-fold each). Disomy 21 was about twice as frequent as disomy 18, and neither showed a preferential segregation with a sex chromosome. Extrapolating across the genome, approximately 18% of sperm carried a numerical abnormality after NOVP treatment of meiotic cells. Induced effects did not persist to 1-2 years after treatment, suggesting that persistent spermatogonial stem cells were not sensitive to NOVP. These findings establish the hypothesis that conception shortly after certain chemotherapies can transiently increase the risks of fathering aneuploid pregnancies that terminate during development or result in the birth of children with major human aneuploidy syndromes.

Many human immunodeficiency virus (HIV)-infected persons receive prolonged treatment with DNA-reactive antiretroviral drugs. A prospective study was conducted of 26 HIV-infected men who provided samples before treatment and at multiple times after beginning treatment, to investigate effects of antiretrovirals on lymphocyte and sperm chromosomes and semen quality. Several antiretroviral regimens, all including a nucleoside component, were used. Lymphocyte metaphase analysis and sperm fluorescence in situ hybridization were used for cytogenetic studies. Semen analyses included conventional parameters (volume, concentration, viability, motility, and morphology). No significant effects on cytogenetic parameters, semen volume, or sperm concentration were detected. However, there were significant improvements in sperm motility for men with study entry CD4 cell counts >200 cells/mm(3), sperm morphology for men with entry CD4 cell counts < or =200 cells/mm(3), and the percentage of viable sperm in both groups. These findings suggest that nucleoside-containing antiretrovirals administered via recommended protocols do not induce chromosomal changes in lymphocytes or sperm but may produce improvements in semen quality.

Etoposide, a topoisomerase II inhibitor widely used in cancer therapy, is suspected of inducing secondary tumors and affecting the genetic constitution of germ cells. A better understanding of the potential heritable risk of etoposide is needed to provide sound genetic counseling to cancer patients treated with this drug in their reproductive years. We used a mouse model to investigate the effects of clinical doses of etoposide on the induction of chromosomal abnormalities in spermatocytes and their transmission to zygotes by using a combination of chromosome painting and 4',6-diamidino-2-phenylindole staining. High frequencies of chromosomal aberrations were detected in spermatocytes within 64 h after treatment when over 30% of the metaphases analyzed had structural aberrations (P < 0.01). Significant increases in the percentages of zygotic metaphases with structural aberrations were found only for matings that sampled treated pachytene (28-fold, P < 0.0001) and preleptotene spermatocytes (13-fold, P < 0.001). Etoposide induced mostly acentric fragments and deletions, types of aberrations expected to result in embryonic lethality, because they represent loss of genetic material. Chromosomal exchanges were rare. Etoposide treatment of pachytene cells induced aneuploidy in both spermatocytes (18-fold, P < 0.01) and zygotes (8-fold, P < 0.05). We know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring. Thus, we found that therapeutic doses of etoposide affect primarily meiotic germ cells, producing unstable structural aberrations and aneuploidy, effects that are transmitted to the progeny. This finding suggests that individuals who undergo chemotherapy with etoposide may be at a higher risk for abnormal reproductive outcomes especially within the 2 months after chemotherapy.

Study Design: Data were collected from 183 subjects who were randomly assigned to the training and test groups. During testing of the classification system, knowledge of the low back pain condition or motion characteristics of the patients in the test group was not made available to the system., Objectives: To determine specific characteristics of trunk motion associated with different categories of spinal disorders and to determine whether a neural network analysis system can be effective in distinguishing patterns., Summary of Background Data: Numerous studies have established the difficulty of evaluating lower back pain. Imaging techniques are expensive and ineffective in many cases. A technique for evaluation of lower back pain was developed on the basis of analysis of such dynamic motion features as shape, velocity, and symmetry of movements, using a neural network classification system., Methods: Dynamic motion data were collected from 183 subjects using a triaxial goniometer. Features of the movement were extracted and provided as input to a two-stage neural network classifier governed by a radial basis function architecture. After training, the output of the classifier was compared with Québec Task Force pain classifications obtained for the patients. Linear and nonlinear classification techniques were compared., Results: The system could determine low back pain classification from motion characteristics. The neural network classifier produced the best results with up to 85% accuracy on novel "validation" data., Conclusions: A neural network based on kinematic data is an excellent predictive model for classification of lower back pain. Such a system could markedly improve the management of lower back pain in the individual patient.

Birth defects cause a myriad of societal problems and place tremendous anguish on the affected individual and his or her family. Current estimates categorize about 3% of all newborn infants as having some form of birth defect or congenital anomaly. As more precise means of detecting subtle anomalies become available this estimate, no doubt, will increase. Even though birth defects have been observed in newborns throughout history, our knowledge about the causes and mechanisms through which these defects are manifested is limited. For example, it has been estimated that around 20% of all birth defects are due to gene mutations, 5-10% to chromosomal abnormalities, and another 5-10% to exposure to a known teratogenic agent or maternal factor [D.A. Beckman, R.L. Brent, Mechanisms of teratogenesis. Ann. Rev. Pharmacol. Toxicol. 24 (1984) 483-500; K. Nelson, L.B. Holmes Malformations due to presumed spontaneous mutations in newborn infants, N. Engl. J. Med. 320 (1989) 19-23.]. Together, these percentages account for only 30-40%, leaving the etiology of more than half of all human birth defects unexplained. It has been speculated that environmental factors account for no more than one-tenth of all congenital anomalies [D.A. Beckman, R.L. Brent, Mechanisms of teratogenesis, Ann. Rev. Pharmacol. Toxicol. 24 (1984) 483-500]. Furthermore, since there is no evidence in humans that the exposure of an individual to any mutagen measurably increases the risk of congenital anomalies in his or her offspring' [J.F. Crow, C. Denniston, Mutation in human populations, Adv. Human Genet. 14 (1985) 59-121; J.M. Friedman, J.E. Polifka, Teratogenic Effects of Drugs: A Resource for Clinicians (TERIS). The John Hopkins University Press, Baltimore, 1994], the mutagenic activity of environmental agents and drugs as a factor in teratogenesis has been given very little attention. Epigenetic activity has also been given only limited consideration as a mechanism for teratogenesis. As new molecular methods are developed for assessing processes associated with teratogenesis, especially those with a genetic or an epigenetic basis, additional environmental factors may be identified. These are especially important because they are potentially preventable. This paper examines the relationships between chemicals identified as human teratogens (agents that cause birth defects) and their mutagenic activity as evaluated in one or more of the established short-term bioassays currently used to measure such damage. Those agents lacking mutagenic activity but with published evidence that they may otherwise alter the expressions or regulate interactions of the genetic material, i.e. exhibit epigenetic activity, have likewise been identified. The information used in making these comparisons comes from the published literature as well as from unpublished data of the U.S. National Toxicology Program (NTP).

Chemicals, by virtue of their varied interactions with biological molecules, are expected to differ in the way they may alter female reproduction. Reproductive toxicity may reflect effects either on the female germ cells or on various maternal processes such as ovulation, implantation, pregnancy, and parturition. In either case, the ultimate manifestation of chemical toxicity on female reproduction is a decrease in the number of normal young born. Very little information is available on the effects of chemicals that are nonhormonal in nature on the long-term ability of treated females to produce offspring. This report presents the results of long-term female total reproductive capacity (TRC) tests on 29 chemicals, including pharmaceuticals, pesticides, and alkylating and industrial agents. For each chemical, the minimum test involved an evaluation of the maximum tolerated dose administered as a single intraperitoneal injection. Females were single-pair mated with an untreated male for most of the female's reproductive life span (a minimum of 347 days posttreatment) and scored for the number of live births produced during this period. Confirmatory dominant lethal experiments or histological examinations for numbers of small follicles were carried out when mutagenic effects or cytotoxicity, respectively, were suspected as the basis for reduced fertility. Of the 29 chemicals studied, 17 had reproductive effects which may be grouped into one of three classes: (1) those that reduced the total number of young and litters per female, (2) those that reduced the total number of young but not of litters, and (3) those that had no significant effect on the total number of young produced but reduced the size of the first and/or second litters. The TRC provides a capacity for detecting a range of toxic insults upon female reproduction. Many of the chemicals were indeed shown to affect the reproductive performance of females through mutagenic and/or cytotoxic effects on follicles. In some cases, however, no causative mechanism could be identified for the observed reduction in reproductive performance. Nevertheless, with this report the number of chemicals tested by this TRC procedure has been quadrupled and the categories of chemicals tested have been substantially broadened.

Multi-color fluorescence in situ hybridization (FISH) was employed to investigate variations in the frequency of aneuploid spermatids produced by males derived from three separate lines of Robertsonian translocations in mice: Rb(2.8)2Lub, Rb(8.12)22Lub, and Rb(8.14)16Rma, each with one arm involving chromosome 8. The DNA probes used were specific for repetitive sequences on chromosomes 8 and X. Heterozygous males for these Robertsonian translocations produced approximately 1% of spermatids with hyperhaploid for chromosome 8. which was > 80 times higher than the frequency of sperm hyperhaploid for chromosome X within the same animals; consistent elevations in chromosome-8 sperm disomy were observed among lines. In addition, approximately 25% higher fractions of sperm aneuploidy were observed when the Robertsonian translocation was inherited from the father rather than from the mother (p = 0.009). These findings illustrate the sensitivity of the FISH procedure for detecting small differences in the hyperhaploidy in male germ cells and suggest that imprinted factors may influence sperm aneuploidy.

The evidence for mammalian germ cell mutagenicity induced by anticancer drugs is summarized. Primary attention is paid to the three major mouse germ cell mutagenicity tests- the dominant lethal, heritable translocation, and morphological specific locus tests- from which most germ cell mutagenicity data historically have been obtained. Of the 21 anticancer drugs reviewed, 16 have been tested in one or more of these three tests; with all 16 tested in the most common germ cell test, the male dominant lethal test, and 9 of the 16 also tested in the female dominant lethal test. The patterns of germ cell stage specificity for most of the anticancer drugs are similar, and generally resemble the patterns seen with other types of chemicals; however, some of the patterns are unique. For example, 2 of the 8 chemicals shown to induce dominant lethal mutations in female oocytes, do not induce dominant lethal mutations in male germ cells (adriamycin and platinol). Ten of the 16 chemicals tested in the dominant lethal test were positive in post-meiotic stages (spermatids through mature sperm), and seven also induced reciprocal translocations and/or specific locus mutations in post-meiotic stages. This propensity to induce mutations in post-meiotic stages has been observed with most mutagens. However, 5 of the anticancer drugs also induced dominant lethal mutations in spermatocytes (meiotic prophase cells) and one of them, 6-mercaptopurine, uniquely induced dominant lethal mutations exclusively in preleptotene spermatocytes. Finally, three of the anticancer drugs (melphalan, mitomycin C, procarbazine) are members of a very select group of chemicals shown to induce specific locus mutations in spermatogonial stem cells of mice. The implications for human risk are discussed.

The chromosomal effects of chloral hydrate (CH) on germ cells of male mice were investigated using two methods to detect and characterize spermatid micronuclei (SMN); (a) anti-kinetochore immunofluorescence (SMN-CREST) and (b) multicolor fluorescence in situ hybridization with DNA probes for centromeric DNA and repetitive sequences on chromosome X (SMN-FISH). B6C3F1 mice received single intraperitoneal (i.p.) injections of 82.7, 165.4, or 413.5 mg/kg and round spermatids were sampled at three time intervals representing cells treated in late meiosis, early meiosis, or as spermatogonial stem cells. No increases in the frequencies of SMN were detected for cells treated during meiosis using either SMN-CREST or SMN-FISH methods. After spermatogonial stem-cell treatment, however, elevated frequencies of SMN were detected by both methods. With SMN-FISH, dose trends were observed both in the frequencies of spermatids containing micronuclei and in the frequency of spermatids carrying centromeric label. These findings corroborate the recent report by Allen and colleagues [Allen JW et al.(1994): Mutat. Res. 323:81-88] that CH treatment of spermatogenic stem cells induced SMN. Furthermore, our findings suggest that chromosomal malsegregation or loss may occur in spermatids long after CH treatment of stem cells. Further studies are needed to understand the mechanism of action of the CH effect on stem cells and to determine whether similar effects are induced in human males treated with CH.

Cyclophosphamide (CP) is used to treat a wide range of neoplastic diseases as well as some non-malignant ones such as rheumatoid arthritis. It is also used as an immunosuppressive agent prior to organ transplantation. CP is, however, a known carcinogen in humans and produces secondary tumors. There is little absorption either orally or intravenously and 10% of the drug is excreted unchanged. CP is activated by hepatic mixed function oxidases and metabolites are delivered to neoplastic cells via the bloodstream. Phosphoramide mustard is thought to be the major anti-neoplastic metabolite of CP while acrolein, which is highly toxic and is produced in equimolar amounts, is thought to be responsible for most of the toxic side effects. DNA adducts have been formed after CP treatment in a variety of in vitro systems as well as in rats and mice using 3H-labeled CP. 32P-postlabeling techniques have also been used in mice. However, monitoring of adducts in humans has not yet been carried out. CP has also been shown to induce unscheduled DNA synthesis in a human cell line. CP has produced mutations in base-pair substituting strains of Salmonella tryphimurium in the presence of metabolic activation, but it has been shown to be negative in the E. coli chromotest. It has also been shown to be positive in Saccharomyces cerevisiae in D7 strain for many endpoints but negative in D62.M for aneuploidy/malsegregation. It has produced positive responses in Drosophila melanogaster for various endpoints and in Anopheles stephensi. In somatic cells, CP has been shown to produce gene mutations, chromosome aberrations, micronuclei and sister chromatid exchanges in a variety of cultured cells in the presence of metabolic activation as well as sister chromatid exchanges without metabolic activation. It has also produced chromosome damage and micronuclei in rats, mice and Chinese hamsters, and gene mutations in the mouse spot test and in the transgenic lacZ construct of Muta Mouse. Increases in chromosome damage and gene mutations have been found in the peripheral blood lymphocytes of nurses, pharmacists and female workers occupationally exposured to CP during its production or distribution. Chromosome aberrations, sister chromatid exchanges and gene mutations have been observed in somatic cells of patients treated therapeutically with CP. In general, there is a maximum dose and an optimum time for the detection of genetic effects because the toxicity associated with high doses of CP will affect cell division. In germ cells, CP has been shown to induce genetic damage in mice, rats and hamsters although the vast majority of such studies have used male mice.(ABSTRACT TRUNCATED AT 400 WORDS)

The zygote and subsequent preimplantation stages of early mammalian development are susceptible to certain chemical perturbations that cause abnormal development of the conceptus. In certain cases, disruption in patterns of gene expression could be a primary event leading to abnormal development. To investigate this hypothesis, we treated pregnant mice with trans-retinoic acid, a known modulator of gene expression. Treatments were administered at various times during pregastrulation stages and the presumed onset of gastrulation. trans-Retinoic acid induced a distinctive set of malformations, as manifest by supernumerary and ectopic limbs and duplication of portions of the lower body, but only when administered during the period of 4.5-5.5 days after mating. (Other malformations were induced at different stages.) The limb and lower-body duplications suggest that exogenous trans-retinoic acid may influence not only the pattern for the hindlimbs but also that for the entire lower body. Since it appears likely that the embryos were affected in the late blastocyst and proamniotic-embryo stages, the provocative possibility arises that aspects of pattern formation of limbs and lower body actually occur prior to gastrulation.

Phosphine (PH3) is a highly toxic grain fumigant to which there is significant human workplace exposure. To determine the in vivo cytogenetic effects of inhalation of PH3, male F344/N rats and B6C3F1 mice were exposed to target concentrations of 0, 1.25, 2.5, or 5 ppm PH3 for 6 hr/day for 9 days over an 11-day period. Approximately 20 hr after the termination of exposures, blood was removed from the mice and rats by cardiac puncture and the lymphocytes cultured for analyses of sister chromatid exchanges and chromosome aberrations in rats and mice, and micronuclei (MN) in cytochalasin B-induced binucleated lymphocytes from mice. In addition, bone marrow (rats) and peripheral blood (mice) smears were made for the analysis of MN in polychromatic and normochromatic erythrocytes. No significant increase in any of the cytogenetic endpoints was found at any of the concentrations examined. These results indicate that concentrations of PH3 up to 5 ppm are not genotoxic to rodents when administered by inhalation for 9 days during an 11-day period as measured by several cytogenetic assays. To evaluate the effects of PH3 on male germ cells, a dominant lethal test was conducted in male mice exposed to 5 ppm PH3 for 10 days over a 12-day period and mated to groups of untreated females (2 females/male) on each of 6 consecutive 4-day mating intervals. None of the 6 groups of females exhibited a significant increase in percent resorptions. These results indicate that exposure to 5 ppm PH3 by inhalation does not induce dominant lethality in male mouse germ cells at steps in spermatogenesis ranging from late differentiating spermatogonia/early primary spermatocytes through mature sperm.

The toxicokinetics of salicylazosulfapyridine (SASP) and its metabolites were investigated in male and female B6C3F1 mice either following single intravenous (5 mg/kg) or oral (67.5, 675, 1350, and 2700 mg/kg) doses, or following three consecutive daily oral doses (675, 1350, and 2700 mg/kg). Plasma concentrations of SASP and its metabolites were quantified by HPLC. Upon intravenous administration, SASP rapidly disappeared from blood with a mean residence time of 0.45-0.78 hr. The only metabolite of SASP found in plasma after an intravenous dose was sulfapyridine (SP). In both sexes, the absolute oral bioavailability of SASP ranged between 16.6-18.2% at a dose of 67.5 mg/kg, and between 2.6-8.7% at doses of 675-2700 mg/kg. Following oral administration of SASP, both SP and AcSP were identified in plasma. The area under the plasma concentration-time curves (AUC) of SP at all four oral doses were approximately 21- to 32-fold or 5- to 25-fold greater than those of SASP in male or female mice, respectively. The acetylated form of SP and AcSP, produced AUC values higher than SASP but much less than SP. Multiple oral doses with SASP did not alter the temporal patterns of SASP absorption and elimination in comparison to a single dose. However, SP accumulated in both sexes following multiple oral doses. A gender-dependent difference in toxicokinetic profiles for SASP and SP was also observed. Female mice displayed a higher Cmax of SASP and SP than did male mice. Although the volume of distribution of SASP was similar in both sexes, the systemic clearance of SASP in males was about twice that observed in females.(ABSTRACT TRUNCATED AT 250 WORDS)

Because genetically based diseases have a major impact on human health, the National Institute of Environmental Health Sciences (NIEHS) has conducted a research and testing program for more than a decade to address chemical induction of heritable genetic damage in the germ cells of mammals. Although most genetic disease results from preexisting mutations, a portion is due to the occurrence of new mutations. The supposition that exposure to mutagenic chemicals contributes to the occurrence of new mutations in the human population is strongly supported by the results from animal models. Such studies clearly demonstrate the potential of environmental chemicals to induce mutations in both somatic and reproductive cells of mammals. This NIEHS program has become a leader in the identification of genetic hazards in the environment and in the acquisition of animal model data used by regulatory agencies in assessing genetic risks to human health.

Limited comparative data in mice indicate that chemical mutagens that induce dominant lethal mutations in males are not necessarily effective in females, but those which are effective in females are generally equally or more effective in males. Recently, however, a few chemicals have been identified that are female-specific with respect to induction of dominant lethal mutations. The antitumor antibiotic adriamycin is among them. Another antitumor antibiotic, bleomycin was examined for its ability to induce dominant lethal mutations in the reproductive cells of male and female mice. No dominant lethal or cytotoxic effects were observed in males treated with bleomycin, even at a maximum tolerated dose. In females, on the other hand, a dose nearly 1/4 of that used in males induced not only a high level of dominant lethal mutations but also killed oocytes in certain stages of follicular development. The effectiveness of bleomycin in inducing dominant lethal mutations in mouse oocytes makes it a valuable tool for investigating whether gonadal transport, inherent differences in the configuration of chromatin in the germ cells of the two sexes or other factors are responsible for the differential susceptibility to bleomycin, which implies potential gender-specific genetic risk in cancer chemotherapy.

The first randomized, blinded, placebo-controlled human trials of recombinant basic fibroblast growth factor (bFGF) for pressure sore treatment were performed. Three different concentrations of bFGF in five dosing schedules were tested for safety using hematology, serum chemistries, urinalysis, absorption, antibody formation, and signs of toxicity. Efficacy was evaluated by wound volumes, histology, and photography. No toxicity, significant serum absorption, or antibody formation occurred. In six of eight subgroups, there was a trend toward efficacy with bFGF treatment. When all subgroups were combined, comparison of the slopes of the regression curves of volume decrease over initial pressure sore volume demonstrated a greater healing effect for the bFGF-treated patients (p < 0.05). Histologically, bFGF-treated wound sections demonstrated increased fibroblasts and capillaries. More patients treated with bFGF achieved > 70% wound closure (p < 0.05). Blinded observers were able to distinguish differences in visual wound improvement between bFGF and placebo groups. These data suggest that bFGF may be effective in the treatment of chronic wounds.

Sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) are the two primary metabolites of the anti-inflammatory drug salicylazosulfapyridine (SASP). These two metabolites were studied for induction of chromosomal damage in mammalian cells, in vitro and in vivo, in an attempt to understand better the genetic effects produced by SASP in humans and laboratory mice. To this end, SP and 5-ASA were tested for induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Abs) in Chinese hamster ovary (CHO) cells in vitro. In addition, they were tested in vivo for induction of micronuclei (MN) in mouse bone marrow polychromatic erythrocytes (PCE). SP gave positive results in the in vitro SCE test and the in vivo MN test, and negative results in the in vitro Abs test. 5-ASA was negative in all three tests. These results indicate that it is the SP metabolite of SASP that is necessary for the induction of chromosomal damage reported to occur in humans and mice after treatment with SASP.

Salicylazosulfapyridine (SASP) and its major metabolite sulfapyridine (SP) have been shown to induce chromosomal damage in vivo. Both chemicals were tested in the micronucleus (MN)/kinetochore (KC) staining test to gain insight into the question of whether chromosomal breakage, aneuploidy-inducing events, or both were important to the observed production of MN in bone marrow cells of mice. In this test, both SASP and SP were shown to be strong inducers of kinetochore positive (KC+) MN. Although small increases in kinetochore negative (KC-) MN were also observed in SP treated mice, as well as in mice receiving the highest dose of SASP tested, the results suggest that both chemicals induce predominantly aneuploidogenic type damage.

In dominant lethal studies the primary variables of interest are typically expressed as discrete counts or proportions (e.g., live implants, resorptions, percent pregnant). Simple statistical sampling models for discrete data such as binomial or Poisson generally do not fit this type of data because of extra-binomial or extra-Poisson departures from variability predicted under these simple models. Extra-variability in the fetal response may originate from parental contributions. These can lead to over- or under-dispersion seen as, e.g., extra-binomial variability in the proportion response. Utilizing a large control database, we investigated the relative impact of extra-variability from male or female contributions on the endpoints of interest. Male-related effects did not seem to contribute to overdispersion in our database; female-related effects were, however, evidenced. Various statistical methods were considered to test for significant treatment differences under these forms of sampling variability. Computer simulations were used to evaluate these methods and to determine which are most appropriate for practical use in the evaluation of dominant lethal data. Our results suggest that distribution-free statistical methods such as a nonparametric permutation test or rank-based tests for trend can be recommended for use.

Chronic wounds such as venous stasis ulcers have become a socioeconomic problem. Even with successful initial management, the recurrence rate approaches 70%. With the advent of new wound healing agents, nonoperative attempts to heal these wounds appear indicated. This study reports a prospective randomized evaluator-blinded trial comparing two potential wound healing agents to an inert vehicle placebo. Eighty-six evaluable patients completed the trial. Silver sulfadiazine 1% in a cream proved to statistically reduce the ulcer size compared with a biologically active tripeptide copper complex 0.4% cream formulation or the placebo. There was no difference between the latter two treatments. Silver sulfadiazine has been shown to allow keratinocyte replication and to have antiinflammatory properties. In this trial its antibacterial action was not used since all ulcers had comparable bacterial levels (less than or equal to 10(5)/gm of tissue) before treatment. These results suggest that the silver sulfadiazine cream used in this study may facilitate healing in wounds healing largely by the process of epithelialization.

Exposure of mouse zygotes to ethylene oxide (EtO) or ethyl methanesulfonate (EMS) led to high incidences of fetal death and of certain classes of fetal malformations (Generoso et al., 1987, 1988; Rutledge and Generoso, 1989). These effects were not associated with induced chromosomal aberrations (Katoh et al., 1989) nor are they likely to be caused by gene mutations (Generoso et al., 1990). Nevertheless, the anomalies observed in these studies resemble the large class of stillbirths and sporadic defects in humans that are of unknown etiology, such as cleft palate, omphalocoel, clubfoot, hydrops and stillbirths (Czeizel, 1985; Oakley, 1986). Therefore, we continue to study the possible mechanisms relating to induction of these types of zygote-derived anomalies in mice. Effects of zygote exposure to the compounds methyl methanesulfonate (MMS), dimethyl sulfate (DMS), and diethyl sulfate (DES), which have similar DNA-binding properties as EtO and EMS, were studied. DMS and DES, but not MMS, induced effects that are similar to those induced by EtO and EMS. Thus, no site-specific alkylation product was identifiable as the critical target for these zygote-derived anomalies. We speculate that the developmental anomalies arose as a result of altered programming of gene expression during embryogenesis.

Salicylazosulfapyridine, commonly known as sulfasalazine or SASP, is an anti-inflammatory drug that is widely used in the treatment of diseases such as ulcerative colitis and Crohn's disease. Increases in sister chromatid exchanges (SCE) and micronuclei (MN) frequencies have been reported in lymphocytes of patients maintained on SASP therapy for up to 21 months. We have tested SASP for its ability to induce chromosome aberrations (ABS) and SCE in cultured Chinese hamster ovary (CHO) cells, ABS in mouse bone marrow cells, and MN in erythrocytes from both bone marrow and peripheral blood of mice. In vitro assays for ABS and SCE were negative. In vivo, SASP administered by single gavage at doses up to 1000 mg/kg did not increase ABS in bone marrow cells of male B6C3F1 mice; however, increases in MN were observed in the peripheral blood erythrocytes of male and female B6C3F1 mice administered 675, 1350 or 2700 mg/kg SASP by gavage for 90 days. Weak but significant dose-related increases in MN were also observed in the bone marrow cells of male B6C3F1 mice administered 500, 1000 and 2000 mg/kg SASP for 3 days. These positive findings in mice support the role of SASP in the induction of MN and SCE in humans, and suggest the need for further evaluation of possible adverse human health effects associated with SASP therapy.

For some chemicals, induction of presumed dominant lethal mutations has been observed only in female mice and not in males. In those cases, questions arise as to (1) whether the increased embryonic mortality is due to genetic effects of the chemicals in the oocyte or, (2) is caused indirectly through maternal toxicity, and, if genetic, (3) the basis for the sex difference. These questions were studied using the compounds adriamycin and platinol. Neither compound induces dominant lethals in male germ cells, but both increased early embryonic mortality when females were treated prior to mating (treatment of maturing oocytes). Reciprocal zygote transfer experiments ruled out, either entirely or for the large part, maternal toxicity as the cause, and cytogenetic analysis of first-cleavage metaphases revealed high incidences of chromosomal aberrations. The results of both of these experiments thus provide evidence that the early embryonic mortality resulted from genetic effects induced in oocytes. Most interestingly, each compound produced unexpected types of chromosomal aberrations. Adriamycin produced deletions, rings, and presumed chromosome-type rearrangements. Platinol, on the other hand, produced a few chromatid-type aberrations, but the bulk of aberrations were characterized by disorganization of the chromatin, minute fragments, and thread-like chromatin bridges between fragments and chromosomes or between two or more chromosomes. The latter type of cytogenetic damage was observed primarily in the centromeric region. It is hypothesized that the female-specific dominant lethal effects of the two compounds are associated with the diffused state of the maturing oocyte chromosomes.

The commercial non-ionic detergent octyl beta-D-glucopyranoside is often contaminated by significant amounts of UV absorbing and/or ionic compounds that can associate with membrane proteins. Such impurities can be monitored by several techniques (i.e., spectrophotometry, size exclusion chromatography, and pH, conductivity, and surface tension measurements) and can be removed using mixed-bed ion exchange chromatography. High performance size exclusion chromatography, dynamic light scattering, and ultracentrifugation have been used to re-estimate the size of micelles of octyl beta-D-glucopyranoside since previously published data varied over a wide range. Aggregation numbers were 27 to 100 for micellar molecular weights 8000 to 29,000. Direct physical methods that do not perturbate the sample indicated a large size for the micelles (hydrodynamic radius 23 +/- 3 A; Mr 22,000 +/- 3000; aggregation number 75 +/- 10 for a 34 mM aqueous solution). In contrast the chromatographic micellar size appeared to be smaller (hydrodynamic radius 15 +/- 1 A; Mr 8000 +/- 1000; aggregation number 27). This underestimation may be the result of adsorption and/or alteration of the micelles.