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The appropriate use of human biomonitoring data to model population chemical exposures is challenging, especially for rapidly metabolized chemicals, such as agricultural chemicals. The objective of this study is to demonstrate a novel approach integrating model predicted dietary exposures and biomonitoring data to potentially inform regulatory risk assessments. We use lambda-cyhalothrin as a case study, and for the same representative U.S. population in the National Health and Nutrition Examination Survey (NHANES), an integrated exposure and pharmacokinetic model predicted exposures are calibrated to measurements of the urinary metabolite 3-phenoxybenzoic acid (3PBA), using an approximate Bayesian computing (ABC) methodology. We demonstrate that the correlation between modeled urinary 3PBA and the NHANES 3PBA measurements more than doubled as ABC thresholding narrowed the acceptable tolerance range for predicted versus observed urinary measurements. The median predicted urinary concentrations were closer to the median measured value using ABC than using current regulatory Monte Carlo methods.

Many patients with moderately to severely active Crohn's disease do not respond to available therapies or lose response over time. The GALAXI-1 study previously found that three intravenous guselkumab dosages showed superior clinical and endoscopic outcomes over placebo at week 12 in patients with moderately to severely active Crohn's disease. We report the safety and efficacy of subcutaneous guselkumab maintenance regimens to week 48 in the GALAXI-1 study.

The relationship between the addition of extra-amniotic saline infusion (EASI) and the efficacy of the Foley catheter in women undergoing cervical ripening and induction of labor with an unfavorable cervical examination is discussed. EASI is unable to improve the efficacy of cervical ripening and induction of labor with a Foley catheter and concurrent oxytocin infusion.

A study was undertaken to develop a model of chronic intrauterine and fetal infection with prevottela bivia, an anaerobe of the lower genital tract that is associated often with bacterial vaginosis. The result indicates that thirty timed pregnant New Zealand White rabbits were inoculated with P bivia solution, this model serve to explore the mechanism of preterm birth that is induced by chronic infection with genital tract anaerobes.

A uterine infection with the bacterium Gardnerella vaginalis causes an increase in tumor necrosis factor alpha in amniotic fluid, according to a study in rabbits. This can damage the fetus and cause low birth weight and premature birth. In addition, this study found the bacteria in the brain and lungs of the fetus.

Antibiotics will not necessarily cure an intrauterine infection, according to a study in rabbits. Even if amniotic fluid is no longer infected, the fetus may still be.

Genetically engineering endometrial cancer cells with the receptor for progesterone can make the cells responsive to the anti-cancer effect of progesterone. Researchers used adenovirus to introduce the gene into endometrial cancer cells in culture.

Background: The nonsteroidal antiestrogen tamoxifen (TAM) is the front-line endocrine treatment for breast cancer, but disease recurrence is common. Treatment failure may occur because tumors become insensitive to TAM. Alternatively, resistance may occur because tumors become stimulated rather than inhibited by TAM. TAM-stimulated growth of MCF-7 human breast tumors has been observed in athymic mice after prolonged treatment with TAM. Purpose: Our purpose was to examine the mechanism of treatment failure by determining whether TAM-stimulated tumors acquire the ability to excrete TAM and its antiestrogenic metabolites or to convert them to estrogenic compounds with weakened antiestrogenic activity. Methods: We used high-pressure liquid chromatography to quantitate TAM and its metabolites in serum and tumors from ovariectomized athymic mice and in MCF-7 cells grown in vitro. We treated tumor-bearing mice with subcutaneous sustained-release preparations of estradiol, TAM, or a nonisomerizable (fixed-ring) analogue and then assessed the activity of these compounds on TAM-inhibited parental MCF-7 tumors and on TAM-stimulated MCF-7 TAM tumors. Results: We found negligible differences in intratumoral TAM levels between TAM-inhibited parental MCF-7 tumors and TAM-stimulated MCF-7 TAM variants. We did not detect metabolite E (Met E), an estrogenic TAM metabolite, in serum or tumors. Using MCF-7 cells in vitro, we determined that the (Z) isomer of Met E, the form directly produced by TAM metabolism, must be present in the cell at a concentration of over 1000 ng/g to overcome growth inhibition by physiological levels of TAM and antiestrogenic metabolites, but the (E) isomer of Met E was effective at 10 ng/g. We reasoned that conversion of Met E from the (Z) (a weak estrogen) to (E) isomer (a potent estrogen) would be required if formation of Met E were responsible for TAM-stimulated growth. However, fixed-ring TAM, which can only form (Z) Met E, was shown to be as capable as TAM of initiating and maintaining antiestrogen-stimulated growth of MCF-7 tumors in athymic mice. Conclusion: Metabolism and isomerization of TAM to estrogenic compounds is not the mechanism of TAM-stimulated growth in our model. Implication: Other potential mechanisms for TAM-stimulated growth, such as estrogen receptor mutation, must be investigated so that effective strategies can be devised to control breast cancer once therapy fails. [J Natl Cancer Inst 85:806-812, 19931

The pyrethroid insecticide, bifenthrin, is frequently measured at concentrations exceeding those that induce acute and chronic toxicity to several invertebrate and fish species residing in the Sacramento-San Joaquin Delta of California. Since the brain is considered to be a significant target for bifenthrin toxicity, juvenile steelhead trout (Oncorhynchus mykiss) were treated with concentrations of bifenthrin found prior to (60 ng/L) and following (120 ng/L) major stormwater runoff events with nontargeted metabolomics used to target transcriptomic alterations in steelhead brains following exposure. Predicted responses were involved in cellular apoptosis and necrosis in steelhead treated with 60 ng/L bifenthrin using the software Ingenuity Pathway Analysis. These responses were predominately driven by decreased levels of acetyl-l-carnitine (ALC), docosahexaenoic acid (DHA), and adenine. Steelhead treated with 120 ng/L bifenthrin had reductions of lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), and increased levels of betaine, which were predicted to induce an inflammatory response. Several genes predicted to be involved in apoptotic (caspase3 and nrf2) and inflammatory (miox) pathways had altered expression following exposure to bifenthrin. There was a significantly increased expression of caspase3 and mioxin fish treated with 120 ng/L bifenthrin with a significant reduction of nrf2 in fish treated with 60 ng/L bifenthrin. These data indicate that bifenthrin may have multiple targets within the brain that affect general neuron viability, function, and signaling potentially through alterations in signaling fatty acids.

Although treatment of Crohn's disease has improved with development of tumour necrosis factor antagonists, fewer than 50% of patients have sustained benefit. Durable maintenance therapy with orally administered alternative treatments remains an unmet need. We aimed to evaluate the effects of ozanimod, an oral agent selectively targeting sphingosine-1-phosphate receptor subtypes 1 and 5, on endoscopic disease activity in Crohn's disease.

Environmental context Pesticides are critical to agriculture and food production but require ecological risk assessments. Although most risk assessments require data from vertebrate animal testing, we have developed an approach to assess risk to fish, birds and mammals using other means. This approach could help to ensure protection of the environment while minimising animal testing. Rationale Recent directives to reduce animal testing have implications for ecological risk assessment, as several vertebrate tests are used to support these assessments. Therefore, a modern approach was devised to address these key knowledge needs without the use of chemical-specific vertebrate testing. Methodology An ecological risk assessment for a novel acetyl-coenzyme A carboxylase (ACCase) inhibitor herbicide was conducted using alternative lines of evidence. For fish, chemical toxicity distributions were constructed to quantify the probability of effects, and these distributions were compared with exposure estimates for a representative use in soybeans. The effect distributions were further refined based on invertebrate toxicity and partitioning behaviour. For birds and mammals, a joint probability curve was constructed by integrating chemical toxicity distributions and Kenaga exposure distributions. Results The lines of evidence presented in this predictive risk assessment suggest the intended use of a new ACCase inhibitor is unlikely to affect fish, birds, or mammals. Exposure was unlikely to exceed effect estimates, regardless of whether they were derived based on chemical-read across, invertebrate toxicity, or partitioning behaviour. Discussion Key knowledge needs for ecological risk assessment can be informed by lines of evidence that do not require animal testing. The present study demonstrates such an approach by comparing predicted exposure and effects, which are expected to be protective. This predictive approach can be extended to other active ingredients and chemical classes, as well as other taxonomic groups of interest. Future research should aim to integrate new approach methods in a predictive risk assessment framework.

A study was conducted to evaluate an intervention designed to divert seventh-, eighth-, and ninth-grade alternative school students from alternative schools' reputation as gateways to the juvenile and criminal justice systems. The possible reasons and solutions for high rates of return to alternative school are also discussed.

Etrasimod, a once-daily, oral, sphingosine 1-phosphate (S1P) receptor modulator that selectively activates S1P receptor subtypes 1, 4, and 5, with no detectable activity on S1P2,3, is in development for the treatment of immune-mediated diseases, including ulcerative colitis. In these two phase 3 trials, we aimed to evaluate the safety and efficacy of etrasimod in adult patients with moderately to severely active ulcerative colitis.

Objective.To provide information on pregnancy outcomes in women receiving certolizumab pegol (CZP).Methods.The UCB Pharma safety database was searched for pregnancies through to September 1, 2014. Reports for maternal and paternal CZP exposure were included and outcomes examined, and data on CZP exposure, pregnancy, comorbidities, and infant events were extracted by 2 independent reviewers. Concomitant medications and disease activity were reviewed for clinical trial patients.Results.Of 625 reported pregnancies, 372 (59.5%) had known outcomes. Paternal exposure pregnancies (n = 33) reported 27 live births, 4 miscarriages, 1 induced abortion, and 1 stillbirth. Maternal exposure pregnancies (n = 339) reported 254 live births, 52 miscarriages, 32 induced abortions, and 1 stillbirth. Almost all reported pregnancies had exposure to CZP in the first trimester, when organogenesis takes place, and a third of them continued the drug into the second and/or third trimesters. The most frequent indications for maternal CZP use were Crohn disease (192/339) and rheumatic diseases (118/339). Twelve cases of congenital malformation and a single neonatal death were reported.Conclusion.Analysis of pregnancy outcomes after exposure to CZP supports previous reports, suggesting a lack of harmful effect of maternal CZP exposure on pregnancy outcomes. However, additional data from a larger number of outcomes after exposure and studies including an unexposed comparison group are required to fully evaluate CZP safety and tolerability in pregnancy.

Background: Athletes are known to use over-the-counter pain medication. However, the frequency of such use among National Collegiate Athletic Association (NCAA) Division I-A football athletes is unknown.Hypothesis: NCAA Division I-A football athletes who use nonprescription analgesics for pain misuse these medications. Study Design: Cross-sectional study.Methods: The football players (N, 144) who met the criteria and agreed to participate were from 8 NCAA Division I-A schools. The participants were administered the Over the Counter Drug Screen for Athletes, which measures attitudes toward the use of a spectrum of substances.Results: Among football athletes surveyed who took nonprescription analgesics for football-related pain, 37% reported taking more than the recommended dose. This was slightly higher than the 28% of players who stated they have not taken nonprescription analgesics for football-related pain. Thirty-four percent of all athletes reported using more than the recommended dose of nonprescription analgesics. Athletes who purchased their own nonprescription analgesics communicated poorly regarding nonprescription analgesics use. Those lacking knowledge about nonprescription analgesics and those using nonprescription analgesics in anticipation of pain or to avoid missing a practice or game were most likely to misuse nonprescription analgesics.Conclusion: NCAA Division I-A football athletes who use nonprescription analgesics for athletic competition do not misuse nonprescription analgesics.

Two uncontrolled, multicenter feasibility studies evaluated safety and pilot efficacy of selective granulocyte and monocyte adsorption apheresis (GMA) with the Adacolumn Apheresis System for treatment of moderate-to-severe ulcerative colitis (UC) and Crohn disease (CD) patients refractoryintolerant to conventional pharmacologic therapy.

Objective:Derivatives of progesterone, progestins, are used to treat endometrial cancer; however, the pathways activated by the hormone have not been fully investigated. Progesterone acts through two receptor isoforms, progesterone receptors A and B (PRA and PRB), transcription factors that control the expression of downstream genes leading to endometrial differentiation. The purpose of this study was to perform an expression analysis to identify the mechanisms underlying progesterone's growth suppressive and immunomodulatory effects in endometrial cancer.Methods:To study the molecular effects of progesterone, PRs were introduced into Hec50co cells. Expression array analyses followed by confirmatory semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) experiments were performed.Results:Expression analysis demonstrated a significant effect of progesterone after 12 hours of treatment on a number of genes, including cell signaling, DNA remodeling, apoptotic, tumor-suppressor, and transcription factors. Of particular interest was the consistent modulation of cytokines, which generally predicted for a powerful anti-inflammatory effect of progesterone through PR. Specifically, pro-inflammatory genes such as TNFα, IL-1β, and MCP-1/MCAF-1 were down-regulated and anti-inflammatory genes such as TRAP1 and SMAD4 were induced.Conclusion:We have discovered that progesterone has a modulatory effect on inflammation and many other important cellular functions. These effects likely underlie the inhibitory effects of progesterone on tumor growth and invasion.

Hereditary renal cell carcinoma (RCC) in Eker rats results from an inherited insertional mutation in the Tsc2 tumor suppressor gene and provides a valuable experimental model to characterize the function of the Tsc2 gene product, tuberin in vivo. The Tsc2 mutation predisposes the Eker rat to develop renal tumors at an early age. The exact mechanism of Tsc2 mediated tumor suppression is not known, however, there is evidence that it is most likely mediated by changes in cell cycle regulation via the PI3K/Akt pathway. The present study was designed to identify if gene expression was different in Tsc2 heterozygous mutant rat kidney compared to wild-type and if any of those differences are associated with tumorigenesis. cDNA microarray analysis of the untreated Tsc2 (+/−) mutant Long Evans (Eker) rat was compared to the Tsc2 (+/+) wild-type Long Evans rat to search for patterns that might be indicative of the intrinsic role of Tsc2 . Of 4395 genes queried, 3.2% were significantly altered in kidneys from heterozygous mutant rats, of which 110 (76%) were up-regulated and 34 (24%) were down-regulated relative to the wild-type. The genes with altered expression belonged to the functional categories of cell cycle regulation, cell proliferation, cell adhesion and endocytosis. Many of these genes appear to be directly or indirectly regulated by the PI3K/Akt pathway. In addition to the PI3K/Akt pathway, other signaling pathways were also differentially expressed in Tsc2 mutant Eker rat kidneys compared to wild-type rats. The gene expression profiles of the Tsc2 heterozygous mutant and wild-type animals highlights new pathways for investigation that may be associated with the tumorigenic activity of tuberin loss and correlate with the enhanced susceptibility of the Tsc2 mutant animal's tendency to develop renal cell carcinoma.

This article compares the trends in living arrangements of older people in several European countries and in the United States. Trends and cross-country variability in several factors that could account for these cross-national differences, including marital status, fertility, labour force participation and attitudes, are also examined. In most countries the proportion of older people living alone increased substantially between 1970 and 1990. However the increase in living alone stabilised or even declined between 1990 and 2000 in most of the countries analysed indicating a possible reversal in the trend. Increases in proportions of older women who are married and reductions in the proportions childless may partially explain this. Considerable variability in both trends and levels of older people's living arrangements was seen especially between north-western and southern European countries. These variations mirrored contrasts in attitudes towards residential care and parent-child coresidence between the countries. [ABSTRACT FROM AUTHOR]

The PR-A and PR-B isoforms of progesterone receptors (PR) have different physiological functions, and their ratio varies widely in breast cancers. To determine whether the two PR regulate different genes, we used human breast cancer cell lines engineered to express one or the other isoform. Cells were treated with progesterone in triplicate, time-separated experiments, allowing statistical analyses of microarray gene expression data. Of 94 progesterone-regulated genes, 65 are uniquely regulated by PR-B, 4 uniquely by PR-A, and only 25 by both. Almost half the genes encode proteins that are membrane-bound or involved in membrane-initiated signaling. We also find an important set of progesterone-regulated genes involved in mammary gland development and/or implicated in breast cancer. This first, large scale study of PR gene regulation has important implications for the measurement of PR in breast cancers and for the many clinical uses of synthetic progestins. It suggests that it is important to distinguish between the two isoforms in breast cancers and that isoform-specific genes can be used to screen for ligands that selectively modulate the activity of PR-A or PR-B. Additionally, use of natural target genes, rather than "consensus" response elements, for transcription studies should improve our understanding of steroid hormone action.

AbstractPopulation aging and changing family patterns have made elder care an important issue. In 1994, German lawmakers enacted a major reform in the country's long-term care policy, the Dependency Insurance Act (DIA). How, and in what way, will the relative use of formal and informal long-term care services change in response? We address this question using longitudinal data from the German Socio-Economic Panel (GSOEP) to examine the mix of care providers used by older Germans prior to enactment. We find that formal care is more likely to be used by those in the poorest health, the single, or the childless. The presence of daughters increases both the use of family and formal care sources. Future work with more recent waves of the GSOEP is needed to see if family care provision is sustained in an environment of universal public long-term care insurance.

Among the closely watched demographic trends of thelate 20th Century is a pronounced drop in fertilityrates throughout much of the world. Italy presents aparticularly interesting case for study: in 1960,Italy's TFR was 2.41, whereas by 1995 it had fallen to1.17. According to United Nations projections, by 2050Italy will be the second oldest country in the world,with 3.4 persons aged 60 or older per person under 15years of age. Besides overall population ageing,another implication of sustained low fertility issmaller families and kin groups. We investigate theconsequences of projected changes in Italy's birth anddeath rates on the composition of kin groups usingmicrosimulation techniques. Using a startingpopulation taken from the 1994 ``Indagine Multiscoposulle Famiglie'' survey, and projected rates ofmortality and fertility by age and parity produced bythe Italian Institute of Statistics, we simulate thepath of kin-group patterns in Italy during the period1994–2050. While we reproduce the aggregate populationpatterns found in official projections, we conduct ourestimates at the ``micro'' level, keeping track of therelationships between individuals that underlie kingroup patterns. We show the effects of the demographictrends on the existence of daughters and sons forolder mothers, on the number of sisters and brotherswith whom an adult woman could share theresponsibilities of caring an elderly mother, and theeffect of the joint action of the increase inlongevity and the mean age at fertility on theproportion of adult women with a living mother.

Breast cancer is the most common malignancy occurring in Western women, and is one of the leading causes of cancer mortality. The nonsteroidal antiestrogen tamoxifen has been shown to be an effective treatment for pre and postmenopausal women with all stages of the disease. Tamoxifen provides effective palliation when used to treat patients with advanced disease, and adjuvant tamoxifen therapy produces significant increases in both disease-free and overall survival (Early Breast Cancer Trialists Collaborative Group. Lancet 339:1-15, 71-85, 1992). Data from the laboratory have shown that the primary action of tamoxifen is tumoristatic rather than tumoricidal, and long-term therapy is therefore recommended. Unfortunately, many patients experience disease progression while taking tamoxifen. Some tamoxifen resistant tumors may remain sensitive to alternative endocrine therapies, while others may become refractory to any hormonal manipulation. Many models have been developedin vitro andin vivo to study the progression of breast cancer growth from tamoxifen sensitive to tamoxifen resistant. We and others have used long-term estrogen deprivation and long-term tamoxifen exposure to develop cell lines and tumors capable of growth in the presence of clinically relevant tamoxifen concentrations. Recently our laboratory has also shown that mutations in the estrogen receptor can cause an antiestrogen-occupied receptor to behave as though it were occupied by an estrogen. Breast cancer is a highly heterogeneous disease and it is likely that the mechanisms which cause tamoxifen resistant growth are equally heterogeneous. Several of the models from our laboratory and others which may contribute to an understanding of this complex phenomenon are discussed here.

Measurements of the estrogen receptor (ER) and the estrogen-induced progesterone receptor (PgR) are used by most clinicians as indicators of both overall prognosis and likelihood of response to endocrine therapy. Patients with ER+/PgR+ tumors have the highest likelihood of response; conversely, patients with ER-/PgR- tumors have the lowest likelihood of response. Unfortunately, most patients treated successfully with endocrine therapy eventually develop endocrine-resistant disease recurrence. In an effort to study potential mechanisms of endocrine resistance, we have studied discordant ER-/PgR+ tumors, in which the normally estrogen-regulated PgR gene is induced in the apparent absence of ER. Our laboratory has previously cloned, from ER-/PgR+ tumors, a variant ER mRNA precisely missing the sequence corresponding to ER exon 5, and has demonstrated that the truncated protein product translated from this variant RNA is capable of constitutively inducing the expression of an estrogen-responsive reporter gene in a yeast expression vector system (Fuqua et al, Cancer Res 51:105-109, 1991). In the present report we describe further experiments to characterize the activity and biological consequences of expression of this variant ER in human breast cancer cells. We have stably transfected MCF-7 human breast cancer cells with a mammalian expression vector for the exon 5 deletion variant ER. These transfected cells produce a truncated ER protein of the expected 40 kDa size. Cells expressing the exon 5 ER deletion variant constitutively express PgR, and manifest increased anchorage-independent colony formation in the absence of estrogen. Furthermore, the anchorage-dependent growth of these cells was not inhibited by the triphenylethylene antiestrogens tamoxifen or 4-hydroxytamoxifen, unlike MCF-7 cells transfected with a control plasmid, which were growth inhibited by both of these compounds. Interestingly, the pure antiestrogen ICI 164,384 did inhibit the growth of exon 5 ER deletion variant-expressing transfectants. The implications of these results with regard to the treatment of tamoxifen-resistant disease are discussed.

The Toxicity of 1-Week Exposures to Inhaled Chloroform in Female B6C3F1Mice and Male F-344 Rats. Larson, J. L., Wolf, D. C., Morgan, K. T., Mery, S., and Butterworth, B. E. (1994). Fundam. Appl. Toxicol.22, 431-446.

Acute Hepatotoxic and Nephrotoxic Effects of Chloroform in Male F-344 Rats and Female B6C3F1 Mice. Larson, J. L., Wolf, D.C., and Butter Worth B. E. (1993). Fundam. Appl. Toxicol. 20, 302-315. Previous studies demonstrated that chloroform given by oral garage in corn oil caused an increased incidence of liver tumors in male and female mice and kidney tumors in male rats, while administration in drinking water resulted in an increased tumor incidence only in the kidneys of the male rats. The tumorigenicity of this nongenotoxic agent has been postulated to be linked with cytolethality and cell proliferation. This study examined the organ-specific toxicity of acute doses of chloroform. Male F-344 rats were given chloroform by gavage in corn oil at the bioassay closes of chloroform of 0 and 180 mg/kg body wt as well as 34 and1477 mg/kg and necropsied 24 hr later. Additional rats were given a single dose of 180 mg chloroform/kg and administered bromodeoxyuridine (BRDU) 2 hr prior to necropsy at 0.5, 1, 2, 4, and 8 days after chloroform treatment. Female B6C3F1 mice were given chloroform by gavage at the bioassay doses of 0.238, and 477 mg/kg as well/as 34 mg/kg and necropsied at 24 hr after treatment. Additional mice were given a single dose of 350 mg chloroform/kg, labeled with BRDU, and necropsied at 0.5, 1, 2, 4, and 8 days after treatment. The kidneys of male rats administered 34, 180, and 477 mg chloroform/kg exhibited mild to severe proximal tubular necrosis in a dose-dependent manner. A 20-fold increase in the labeling index (LI, the percentage of nuclei in S-phase) in the proximal tubule cells was observed 2 days after treatment with the bioassay dose of 180 mg/kg. The livers of male rats exhibited only slight to moderate multifocal centrilobular necrosis at 180 and 477 mg/kg. A 10-fold increase in the LI was observed in the liver of male rats given 477 mg/kg, but no increase was observed at the bioassay dose of 180 mg/kg. In contrast to male rats, female mice developed a dose-dependent centrilobular hepatic necrosis at 238 and 477 mg/kg. No renal lesions were observed in female mice at any dose. A peak increase in LI of 38-fold was observed in hepatocytes in the livers of female mice 2 days after treatment with 350 mg chloroform/kg, with only a 2 fold increase in LI observed in the kidneys. These data indicate that acute chloroform-induced cytolethality leads to increased cell proliferation and that the organ-specific pattern of toxicity is the same as the organ-specific pattern of tumor formation. These observations are suggestive of a role for induced cell proliferation in tumor formation and indicate that more extensive cell proliferation studies are warranted. Copyright 1993, 1999 Academic Press

Elemental diets designed for nutritional support in protein-calorie malnutrition are often deficient in choline, a nonessential nutrient. Previously, malnourished patients on these diets were found to be at risk of developing plasma choline deficiency. We have now estimated the prevalence of this deficiency by determining fasting plasma levels of choline among cirrhotic and noncirrhotic malnourished male subjects maintained on regular hospital mixed food or elemental parenteral and enteral formulas. Plasma choline concentrations (μM, average ± SD) were as follows: (i) mixed foods, 11.3 ± 4.3 for cirrhotic (n = 22) and 9.3 ± 2.4 for noncirrhotic (n = 12) patients; (ii) parenteral formula, 5.3 ± 1.6 for cirrhotic (n = 5) and 8.6 ± 5.2 for noncirrhotic (n = 16) subjects; and (iii) enteral formula, 6.1 ± 1.2 for cirrhotic (n = 5) and 11.7 ± 1.9 for noncirrhotic (n = 4) subjects. The level for healthy normal subjects eating mixed foods was 12.0 ± 2.2. The prevalence of plasma choline deficiency, i.e., plasma levels ≥2 SD below the normal average, was as follows: parenteral formula, all cirrhotic and 10 of 16 noncirrhotic subjects; enteral formula, allcirrhotic and noneof the noncirrhotic subjects. The reversibility of choline deficiency was examined in a longitudinal study of three phases involving 10 patients—5 with alcoholic cirrhosis (all on enteral formula); 5 noncirrhotic (1 on enteral and 4 on parenteral formula). During phase 1 (3-day equilibration period; ad libitum regular hospital diet), plasma choline levels were within the normal range for all subjects. During phase 2 (2 wk, choline depletion phase, elemental formulas), choline levels were subnormal in all cirrhotic subjects (5.1 ± 2.0 μM) on enteral formula and all noncirrhotic patients on parenteral formula (5.9 ± 1.3 μM). During phase 3 (2 wk, choline repletion phase, elemental formula + 6 g choline/day), the levels normalized in all patients (cirrhotic 11.4 ± 3.1 μM and noncirrhotic 11.9 ± 3.2 μM). Analyses of abdominal computed tomographic scans and plasma liver chemistries in the cirrhotic subjects during the three phases suggested a correlation between plasma choline deficiency and hepatic steatosis and abnormal liver enzyme levels in some patients. Therefore, choline may be an essential nutrient in malnourished cirrhotic patients and its deficiency may be associated with adverse hepatic effects.

A method for generalizing the multistate, or increment-decrement, life table to include rates which depend upon duration of exposure to risk, as well as upon age, is proposed. The method is built upon the linear approximation, called the linear integration hypothesis, developed mainly by Rogers and his colleagues. Although the use of rates indexed by duration categories leads to a substantial increase in the state space of the model, it is possible to arrange the rates in such a way that matrices to be inverted are no larger than those encountered in the usual multistate life table. In the more general approach it is possible to derive several new summary indices of the life-table cohort's history, such as the mean and median time in current status, at any age. The method is illustrated using a simple four-state marital-status model which has appeared often in the literature; here, rates of divorce and widowhood vary by duration of marriage as well as age.

Induced Cytolethality and Regenerative Cell Proliferation in the Livers and Kidneys of Male B6C3F₁, Mice Given Chloroform by Gavage. Larson, J. L., Wolf, D.C., and Butterworth, B. E. (1994). Fundam. Appl. Toxicol. 23, 537-543. It has been reported that chloroform administered to male B6C3F₁ mice at doses of 138 and 277 mg/kg/day in corn oil by gavage 5 days/week for 2 years resulted in incidences of hepatocellular carcinomas of 36 and 98% relative to an incidence in controls of 6%. Cytotoxicity and regenerative cell proliferation have been implicated in the tumorigenic process for this nongenotoxic compound. Although chloroform is known to be nephrotoxic in the male mouse, no treatment-related increase was observed in the frequency of kidney tumors. To better understand the relationship of these endpoints, this study evaluated chloroform-induced cytotoxicity and cell proliferation in the liver and kidney under conditions of the cancer study. B6C3F₁ mice were administered oral doses of 0, 34, 90, 138, or 277 mg/kg/day of chloroform dissolved in corn oil for 4 days or 5 days/week for 3 weeks. Bromo-2'-deoxyuridine (BrdU) was administered via osmotic pumps implanted 3.5 days prior to necropsy to label cells in S-phase. Cell proliferation was evaluated in tissue sections immunohistochemically as the percentage of cells in S-phase (nuclear labeling index; LI). Mice given 34 and 90 mg/kg/day by gavage had mild degenerative changes in centrilobular hepatocytes after 4 days of treatment, which was absent at 3 weeks. Centrilobular necrosis was observed in mice given 138 or 277 mg/kg chloroform for 4 days, with increased severity of necrosis at 3 weeks. The LI in the livers of mice was increased in a dose-dependent manner at all chloroform doses following 4 days of treatment. The hepatic LI was similarly elevated at 3 weeks in the 277-mg/kg dose group, but had declined in the lower dose groups, and was no longer elevated above controls at 34 or 90 mg/kg/day. Kidneys from mice in all dose groups exhibited a dose-related acute tubular necrosis after 4 days of dosing. After 3 weeks of dosing, regenerating tubules were observed in the lower dose groups, while mice treated with 277 mg/kg had severe nephropathy characterized by degeneration, necrosis, and regeneration affecting all of the proximal tubules. The LI in the proximal tubules was increased at all doses after 4 days of treatment. At the end of 3 weeks of dosing, the LI in the renal cortex had decreased at all dose levels and was no longer significantly different from controls in the 34- and 90-mg/kg/day groups. The sustained increase in LI seen in the livers of mice administered not only the maximum tolerated dose (MTD), but also one-half the MTD is consistent with the hypothesis that events secondary to cytolethality and regenerative cell proliferation play a key role in chloroform-induced mouse liver cancer. The observation of toxicity and acute regenerative cell proliferation in the kidney without subsequent tumor formation may be because the severity of the nephrotoxicity in some way actually interfered with tumor formation. Alternatively, the mouse liver may simply be a more sensitive target organ than the kidney to tumor formation induced through mechanisms secondary to regenerative cell proliferation. Copyright 1994, 1999 Academic Press

Methyltert-butyl ether (MTBE), a fuel additive blended into unleaded gasoline to decrease carbon monoxide emissions, induces renal tumors in male, but not female, rats exposed by inhalation to ≥3000 ppm MTBE. A number of chemicals that induce male rat-specific renal tumors also cause a syndrome unique to male rats referred to as α2u-globulin nephropathy (α2u-N). The objective of the present study was to determine if MTBE induces an α2u-N and renal cell proliferation in male F-344 rats. Male and female F-344 rats were exposed to MTBE vapors of 0, 413, 1516, or 3013 ppm for 6 hr/day for 10 consecutive days. Significant proximal tubule necrosis and protein droplet accumulation were observed in kidneys from male rats exposed to 1516 and 3013 ppm MTBE. Significantly greater labeling indices were observed in all groups of MTBE-exposed male rats. α2u-Globulin immunoreactivity was present in and confined to protein droplets in male rat kidney. A mild dose-related increase in α2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to MTBE, with a statistically significant increase in α2u concentration in male rats exposed to 3013 ppm MTBE. There was a strong positive correlation (r= 0.994) with exposure concentration between cell proliferation and α2u concentration in male rat kidney. No significant differences were observed in female rats for any of these responses. Further analysis of kidney cytosol failed to demonstrate the accumulation of any protein besides α2u in MTBE-exposed male rat kidney. These findings demonstrate that MTBE causes a mild induction of α2u-N and enhanced renal cell proliferation in male, but not female, F-344 rats, suggesting a role for α2u-N in renal tumorigenesis.

Potassium bromate (KBrO3) is a rodent carcinogen and a nephro- and neurotoxicant in humans. KBrO3is used in cosmetics and food products and is a by-product of water disinfection by ozonization. KBrO3is carcinogenic in the rat kidney, thyroid, and mesothelium and is a renal carcinogen in the male mouse. The present study was designed to investigate the relationship of time and dose to bromate-induced tumors in male Fischer 344 (F344) rats and to provide some insight into the development of these tumors. KBrO3was dissolved in drinking water at nominal concentrations of 0, 0.02, 0.1, 0.2, and 0.4 g/L and administered to male F344 rats as the sole water source for 12, 26, 52, 78, or 100 wk. Renal cell tumors were present after 52 wk of treatment only in the high-dose group. Mesotheliomas developed after 52 wk of treatment on the tunica vaginalis. Mesotheliomas were present at sites other than the testicle after 78 wk of treatment, indicating that their origin was the testicular tunic. Thyroid follicular tumors were present as early as 26 wk in 1 rat each from the 0.1- and 0.2-g/L groups. The present study can be used as a basis for the determination of dose-time relationships of tumor development for a better understanding of KBrO3-induced cancer.

Modulations in the positive hepatocyte growth factor, transforming growth factor-alpha (TGF-α) and its receptor epidermal growth factor receptor (EGFR), occur in rat and human liver tumors. The purpose of this study was to determine if TGF-α and EGFR are altered in basophilic and acidophilic preneoplastic and neoplastic liver lesions generated in DEN-initiated mice exposed to a variety of hepatocarcinogens. Female B6C3F1 mice were initiated with N-nitrosodiethylamine (DEN) and treated with hepatocarcinogenic concentrations of unleaded gasoline vapor (2,000 ppm), methyl tertiary butyl ether vapor (7,814 ppm), phenobarbital (500 ppm, diet), or chlordane (25 ppm, diet). Hepatic foci and tumors were identified and evaluated immunohistochemically with antibodies for TGF-α and EGFR. In all treatment groups, basophilic hepatic foci were negative for TGF-α immunoreactivity (554/564, 98%). In contrast, regardless of treatment, acidophilic hepatic foci were immunoreactive for TGF-α (107/108, 99%). There was no significant difference in mean hepatic labeling index as measured by the incorporation of 5-bromo-2'-deoxyuridine between foci immunoreactive and nonimmunoreactive for TGF-α. The incidence of immunoreactivity for TGF-α increased in hepatocellular tumors that were predominantly of the basophilic phenotype. Of basophilic hepatocellular adenomas, 16/81 (20%) were immunoreactive for TGF-α, while 17/29 (59%) of hepatocellular carcinomas stained positive for TGF-α. A similar increased incidence of EGFR immunoreactivity was found in basophilic hepatocellular adenomas (17/67, 25%) and carcinomas (19/28, 68%) relative to basophilic foci (11/367, 3%), suggesting an autocrine mechanism for the development of mouse liver tumors. The increased incidence of TGF-α immunoreactivity in basophilic liver tumors suggests that TGF-α is a marker of tumor progression in mouse liver. Furthermore, TGF-α modulations were dependent on phenotype rather than treatment, indicating inherent differences in the expression of TGF-α in basophilic and acidophilic hepatic lesions.

The development of scientifically sound risk assessments based on mechanistic data will enable society to better allocate scarce resources. Inadequate risk assessments may result in potentially dangerous levels of hazardous chemicals, whereas overly conservative estimates can result in unnecessary loss of products or industries and waste limited resources. Risk models are used to extrapolate from high-dose rodent studies to estimate potential effects in humans at low environmental exposures and determine a virtually safe dose (VSD). When information to the contrary is not available, the linearized multistage (LMS) model, a conservative model that assumes some risk of cancer at any dose, is traditionally employed. In the case of airborne chloroform, the dose at which an increased lifetime cancer risk of 10-6 could be calculated was chosen as the target VSD. Applying the LMS model to the mouse liver tumor data from a corn-oil gavage bioassay yields a VSD of 0.000008 ppm chloroform in the air. The weight of evidence indicates that chloroform is not directly mutagenic but, rather, acts through a nongenotoxic-cytotoxic mode of action. In this case, tumor formation results from events secondary to induced cytolethality and regenerative cell proliferation. Toxicity is not observed in rodents when chloroform is not converted to toxic metabolites at a rate sufficient to kill cells. Thus, tumors would not be anticipated at doses that do not induce cytolethality, contrary to the predictions of the LMS model. Inhalation studies in rodents show no cytolethality or regenerative cell proliferation in mouse liver at a chloroform concentration of 10 ppm as the no observed effect level (NOEL) or below. Using that NOEL and a safety factor approach, one can develop a VSD of 0.01 ppm. Integrating these data into the risk assessment process will yield risk estimates that are appropriate to the route of administration and consistent with the mode of action.

Sequential light microscopic and ultrastructural examination of kidneys from male and light microscopic examination of female Mongolian gerbils given 250 mg 2-bromoethylamine hydrobromide (BEA)/kg body weight ip were performed. In addition, male Mongolian gerbils were treated with both BEA and ip injections of either water, dimethyl sulfoxide, piperonyl butoxide, or reserpine. Light microscopic renal lesions in male Mongolian gerbils progressed from congestion of the vasa recta of the proximal inner medulla at 6 hr post-treatment to total renal papillary necrosis (RPN) at 24 hr post-treatment. There was no sex difference in sensitivity to BEA. Ultrastructural alterations in male gerbils were restricted to the vasa recta. Vascular lesions of endothelial swelling and pericapillary edema in the vasa recta of the proximal inner medulla was observed 2 hr post-treatment and progressed to occlusion by platelets adherent to exposed basement membranes at 6 hr post-treatment. Diuresis induced by injections of saline and injections of dimethyl sulfoxide or piperonyl butoxide did not affect the development of BEA-induced RPN. Reserpine slowed the development of BEA-induced RPN by its vasodilatory effect on the renal vasculature, not by blocking the endothelial toxicity of BEA. RPN induced by BEA in the Mongolian gerbil is apparently an ischemic necrosis of the inner medulla that develops secondary to endothelial damage of the vasa recta.

Household composition of older unmarried women in Hungary is analyzed using data from the 1984 microcensus. The principal determinants of household composition investigated are kin availability — the number of living children, siblings, and parents — health status, marital status, age, and income. A multinomial logit model distinguishing among five household types reveals that number of children, severe disabilities, age and income are all strongly related to household composition. Trends in fertility and mortality patterns suggest that kinship patterns will change in coming years; these results imply that household composition will, in turn, change as well.

Induced Cytotoxicity and Cell Proliferation in the Hepatocarcinogenicity of Chloroform in Female B6C3F1Mice: Comparison of Administration by Gavage in Corn Oil vs ad Libitumin Drinking Water. Larson, J. L., Wolf, D. C., and Butterworth, B. E. (1994). Fundam. Appl. Toxicol.22, 90-102.

Liver biopsy with measurement of hepatic iron concentration is the most certain procedure for evaluation of iron-storage disease, although use of computed tomography and magnetic resonance imaging procedures recently have been proposed as alternative, noninvasive methods for estimating the degree of iron overload. The results of these imaging procedures were compared with those of other noninvasive techniques and liver biopsies in 48 patients. Final diagnoses, based on synthesis of clinical and laboratory data, included (a) primary hemochromatosis (n = 25; 19 homozygous, 6 heterozygous); (b) secondary hemochromatosis (n = 7); (c) alcoholic liver disease (n = 11); (d) chronic active hepatitis (n = 3); and (e) other (n = 2). Serum ferritin and computed tomography or magnetic resonance scanning had 100% sensitivity in detecting hepatic iron overload more than fivefold above the upper limit of normal (>10.7 μmol Fe/100 mg dry liver) but did not detect lesser degrees of iron overload reliably, including those found in 6 of 13 patients with untreated homozygous primary hemochromatosis and 3 of 7 with secondary hemochromatosis. Computed tomography and magnetic resonance imaging were more specific than ferritin (64% and 92% vs. 21%) in the detection of iron excess, more than five times the upper limit of normal. Among magnetic resonance imaging measures, the ratio of the second echo signal intensities of liver to paraspinous muscle was the most sensitive and most specific for detection of this degree of iron overload. The degree of correlation between hepatic iron concentration and results of noninvasive laboratory or imaging studies were insufficient to permit prediction of hepatic iron content by noninvasive studies alone. It is concluded that computed tomography or magnetic resonance scanning as currently usually used is not cost-effective in routine evaluation of iron overload, although these imaging procedures may play a role in patients in whom liver biopsy is contraindicated. Because of their low cost and ready availability, serum ferritin and transferrin saturation tests remain the preferred screening studies for iron overload. Liver biopsy with quantitative iron measurement remains the study of choice for the definitive diagnosis of hemochromatosis.

Ozone has been proposed for water disinfection because it is more efficient than chlorine for killing microbes and results in much lower levels of carcinogenic trihalomethanes than does chlorination. Ozone leads to formation of hypobromous acid in surface waters with high bromine content and forms brominated organic by-products and bromate. The carcinogenicity and chronic toxicity of potassium bromate (KBrO3) was studied in male B6C3F1mice and F344/N rats to confirm and extend the results of previous work. Mice were treated with 0, 0.08, 0.4, or 0.8 g/L KBrO3in the drinking water for up to 100 wk, and rats were provided with 0, 0.02, 0.1, 0.2, or 0.4 g/L KBr03. Animals were euthanatized, necropsied, and subjected to a complete macroscopic examination. Selected tissues and gross lesions were processed by routine methods for light microscopic examination. The present study showed that KBr03is carcinogenic in the rat kidney, thyroid, and mesothelium and is a renal carcinogen in the male mouse. KBr03was carcinogenic in rodents at water concentrations as low as 0.02 g/L (20 ppm; 1.5 mg/kg/day). These data can be used to estimate the human health risk that would be associated with changing from chlorination to ozonation for disinfection of drinking water.

As in many other industrialized countries, population aging in Italy is disproportionately a phenomenon associated with unmarried women, mainly widows. This article examines the extent to which older unmarried women live alone, and the extent to which they receive help in everyday tasks from others outside their households, using data from a large Italian household sample survey conducted in 1983. Older women can either live alone or with others, and may or may not receive external help in either case; thus there are four distinct combinations of outcomes analyzed. In both descriptive bivariate analysis. and a multivariate model of the outcomes, we find pronounced differences in behavior according to region of residence, educational level, age, degree of disability, work experience, and pension receipt. The findings indicate the importance of family as a source of help and/or co-residence in situations of need.

The non-steroidal antiestrogen tamoxifen (TAM) is successfully used to treat all stages of breast cancer in both pre- and postmenopausal women. Unfortunately, most women treated with TAM eventually develop resistant tumor recurrences which require intervention with a second-line endocrine therapy, or cytotoxic chemotherapy if the recurrence is completely endocrine insensitive. There is evidence that some recurrences may in fact be TAM stimulated. MCF-7 human breast cancer cells grown as solid tumors in athymic mice chronically treated with TAM reproducibly develop a TAM stimulated phenotype (Osborneet al., Eur J Cancer Clin Oncol 23: 1189-1196, 1987; Gottardis and Jordan, Cancer Res 48: 5183-5187, 1988; Osborneet al., J Natl Cancer Inst 83: 1477-1482, 1991; Wolfet al., J Natl Cancer Inst 85: 806-812, 1993). Tumors of this type may provide a useful model for a subset of therapeutic failures in the clinic. Therefore, we have extensively studied this model in an attempt to define the mechanism or mechanisms leading to TAM stimulated growth. In this paper we describe the characteristics of 4 TAM stimulated MCF-7 tumor variants. All of these tumors are growth stimulated by TAM, but vary in their response to estradiol (E2) treatment, and grow poorly in placebo treated hosts. All tumor variants express estrogen receptor (ER) RNA and protein, which at the RNA level appear to be down regulated by TAM, and to a greater extent by E2. All tumors also express epidermal growth factor receptor (EGFR) RNA, which is down regulated by TAM, and further down regulated by E2. However, among the tumor variants analyzed, ER and EGFR levels appear to be inversely related. Further, despite the expression of ER by all 4 TAM stimulated tumor variants, E2 induction of progesterone receptor expression is very weak or entirely absent.

The nonsteroidal antiestrogen tamoxifen (TAM) is the most commonly used endocrine treatment for all stages of breast cancer in both pre- and postmenopausal women. However, the development of resistance to the drug is common, as most patients treated with TAM eventually experience a recurrence of tumor growth. One of the potential mechanisms of treatment failure is the acquisition by the tumor of the ability to respond to TAM as a stimulatory rather than inhibitory ligand. We (Gottardis and Jordan, Cancer Res 48: 5183-5187, 1988; Wolfet al., J Natl Cancer Inst 85: 806-812, 1993) and others (Osborneet al., Eur J Cancer Clin Oncol 23: 1189-1196, 1987; Osborneet al., J Natl Cancer Inst 83: 1477-1482, 1991) have extensively described the reproducible development of TAM stimulated growth in a laboratory model system using MCF-7 human breast cancer cells grown as solid tumors in athymic mice. In this paper we report on the isolation of an estrogen receptor (ER) from a TAM stimulated tumor (MCF-7/MT2) which contains a point mutation that causes a tyrosine for aspartate substitution at amino acid 351 in the ligand binding domain. The mutant appears to the major form of ER expressed by this tumor. We also report that only wild type ER was detected in three other TAM stimulated MCF-7 tumor variants, suggesting that multiple mechanisms are possible for the development of TAM stimulated growth. The implications of these findings are discussed.

To investigate the incidence of acute complications occurring within 14 days of large loop excision of the transformation zone (LLETZ), the authors reviewed all LLETZ procedures performed at their institution between 1999 and 2001 and identified 557 patients (of a total 567) suitable for analysis. Clinical and demographic information were abstracted from the chart. Almost all procedures were performed by the same physician who adjusted the diameter of the loop for each case, most commonly 10 × 10 mm, maximum 12 mm. A top hat using a 5 × 5-mm loop was performed for every procedure. Fifty percent of patients were younger than 30 years of age, and the majority (75) were younger than 40 years of age. Fifty-nine percent were Hispanic, 21 were white, and 16 were black. Histologic examination of the surgical specimen identified invasive cancer in 4 patients, microinvasive cancer in 23, and cervical intraepithelial neoplasia 3, 2, or 1 in 526 patients (n 321, 150, and 55, respectively). No disease was seen in 4 patients. In all, the complication rate in the first 2 weeks after LLETZ was 9.7. Three of the 557 patients had a major complication. Two patients required surgical repair, 1 for a small bowel injury and 1 for intractable vaginal bleeding. A third patient developed heart palpitations and was placed under observation in the emergency room. Minor complications (defined as problems that required a return clinic visit) included abdominal pain (n 14), vaginal bleeding (n 26), vaginal discharge (n 6), and 1 patient with bladder spasms. Over 90 of the study group returned for follow up.

PS-6 unleaded gasoline (UG) and methyl tert-butyl ether (MTBE), an UG additive, with long-term exposure at high concentrations increased liver tumors selectively in female mice. PS-6 UG is a liver tumor promoter in N-nitrosodiethylamine-initiated female mice and produces short-term effects potentially relevant to its tumor promoting ability. The new formulation of UG (91-01) and MTBE were evaluated for similar short-term effects in mouse liver. Mice were exposed to 7814 ppm MTBE, 2014 ppm 91-01 UG, or 2028 ppm PS-6 UG vapor for 3 or 21 days, 6 hr/day, 5 days/week. Relative liver weights increased and uterine weights decreased in MTBE-, 91-01 UG-, and PS-6 UG-exposed mice. Because the decrease in relative uterine weight is suggestive of hormonal modulation, we evaluated the effects of MTBE, 91- 01 UG, and PS-6 UG in vivo on hepatic 17-β estradiol metabolism in vitro. Gavage treatment with either blend of UG and with MTBE increased estrogen metabolism in isolated mouse hepatocytes. Hepatic microsomal P450 activity was assessed by 7-pentoxyresorufin-O-dealkylase (PROD) and 7-ethoxyresorufin-O-deethylase (EROD) activities. Similar increases in P450 content and PROD and EROD activities were observed in all exposed mice as compared to controls. No hepatoxicity was observed in any treatment group. The hepatic labeling index, as measured by the incorporation of 5-bromo-2′-deoxyuridine, was increased in all exposed mice at 3 days but not 21 days, indicating that MTBE and 91-01 UG are also hepatic mitogens. These data demonstrate that a newer blend of UG and the UG additive MTBE elicit short-term effects similar to those of PS-6 UG. Given that these effects are potentially related to tumor promotion and the general lack of genotoxic activity, MTBE and 91-01 UG may exhibit tumor promoting activity similar to that seen with PS-6 UG. Since the liver is under multihormonal control, the increase in hepatic estrogen metabolism and uterine effects support a potential role for endocrine modulations in both MTBE- and UG-induced hepatocarcinogenesis.

Alterations in the amino acid sequence of the estrogen receptor (ER) have been shown to have dramatic effects on its function. Recently, mutant ERs have been isolated from both clinical samples and established breast cancer cell lines, primarily through the use of the polymerase chain reaction (PCR). All previously reported mutations have given rise to either alterations or truncations of the ER protein. We determined the structure of a novel 80 kDa ER which is expressed in an estrogen independent subclone of the MCF-7 human breast cancer cell line (MCF-7:2A). This 80 kDa ER was initially detected by Western blot analysis using a variety of ER specific antibodies. PCR mapping and partial PCR mediated subcloning of the ER cDNA were used to demonstrate that this protein was an ER containing an in-frame duplication of exons 6 and 7. This type of duplication has not been previously described for any members of the steroid receptor superfamily. Karyotype analysis coupled with fluorescence in situ hybridization (FISH) demonstrated that MCF-7:2A cells contained 4–5 copies of the ER gene in contrast to 2 copies in MCF-7:WS8 cells. The ER gene was localized by FISH analyses in both the MCF-7:WS8 and MCF-7:2A cells on chromosome 6, which is the source of the ER in normal human cells. The relative expression level of 2:1 is consistent with DNA gene dosage analysis. Genomic PCR was then used to demonstrate that the 80 kDa ER mRNA was not derived from the trans-splicing of two ER mRNAs but was the result of a genomic rearrangement in which exons 6 and 7 were duplicated in an in-frame fashion. This variant ER may prove to be useful in elucidating the mechanism of estrogen action in breast cancer cells.

High doses of chloroform induced liver cancer in male and female B6C3F1 mice when administered by gavage, kidney cancer in male Osborne-Mendel rats when given by gavage or in the drinking water, and kidney cancer in male BDF1 mice when administered by inhalation. The weight of evidence indicates that chloroform is acting through a nongenotoxic-cytotoxic mode of action. The present study was designed to investigate the dose-response relationships for chloroform-induced lesions and regenerative cell proliferation in B6C3F1 mice as the basis for formulation of a biologically based risk assessment for inhaled chloroform. Different groups of female and male B6C3F1 mice were exposed to atmospheric concentrations of 0, 0.3, 2, 10, 30, and 90 ppm chloroform 6 hr/day, 7 days/week for exposure periods of 4 days or 3, 6, or 13 consecutive weeks. Some additional exposure groups were exposed for 5 days/week for 13 weeks or were exposed for 6 weeks and then examined at 13 weeks. Bromodeoxyuridine was administered via osmotic pumps implanted 3.5 days prior to necropsy, and the labeling index (LI, percentage of nuclei in S-phase) was evaluated iminunohistochemically from histological sections. Complete necropsy and microscopic evaluation revealed treatment-induced dose- and time-dependent lesions only in the livers and nasal passages of the female and male mice and in the kidneys of the male mice. Large, sustained increases in the liver LI were seen in the 90-ppm groups at all time points. The female mice were most sensitive, with a no-observed-adverse-effect level (NOAEL) for induced hepatic cell proliferation of 10 ppm. The hepatic LI in the 5 days/week groups were about half of those seen in the 7 days/week groups and had returned to the normal baseline in the 6-week recovery groups. Induced renal histologic changes and regenerative cell proliferation were seen in the male mice at 30 and 90 ppm with 7 days/week exposures and also at 10 ppm with the 5 days/week regimen. Nasal lesions were transient and confined to mice exposed to 10, 30, or 90 ppm for 4 days. In a previous cancer bioassay, a gavage dose of 477 mg/kg/day pro duced a 95% liver tumor incidence in female B6C3F1 mice. This gavage dose is equivalent to a daily 6 hr/day inhalation exposure of approximately 80 ppm, based on the observed induced increases in the LI as an internal dosimeter. The United States Enviromnental Protection Agency currently uses the linearized multistage model applied to the mouse liver tumor data from the chloroform gavage study to estimate a virtually safe dose (VSD) as a one in a million increased lifetime risk of cancer. The resulting value is an airborne exposure concentration of 0.000008 ppm. Assuming that chloroform-induced female mouse liver cancer is secondary to events associated with necrosis and regenerative cell proliferation, then no increases in liver cancer in female mice would be predicted at the NOAEL of 10 ppm or below based on the results reported here. Applying an uncertainty factor of 1000 yields an estimate of a VSD at 0.01 ppm. This estimate relies on inhalation data and is more consistent with the mode of action of chloroform.

Detailed quantitative descriptions of the toxicity of inhaled chloroform are lacking, despite the fact that the majority of environmental exposures occur by this route. We investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the livers and kidneys, the principal target organs for carcinogenicity of female B6C3F1 mice and male F-344 rats, respectively. Nasal passages were also examined for toxic responses. Rodents were exposed to chloroform vapors at concentrations of 0, 1, 3, 10, 30, 100, or 300 ppm for 6 hr/day for 7 consecutive days and necropsied on Day 8. Animals were administered bromodeoxyuridine (BrdU) via implanted osmotic pump for the previous 3.5 days before necropsy. Cell proliferation was quantitated as the percentage of cells in S-phase (labeling index; LI) measured by immunohistochemical detection of BrdU-labeled nuclei. Mice exposed to 100 or 300 ppm exhibited centrilobular hepatocyte necrosis and severe vacuolar degeneration of midzonal and periportal hepatocytes, while exposure to 10 or 30 ppm resulted in mild to moderate vacuolar changes in centrilobular hepatocytes. Slight, dose-related increases in the hepatocyte LI were observed for exposure concentrations of 10 and 30 ppm, while the LI was increased more than 30-fold in the 100 and 300 ppm groups. The kidneys of mice were affected only at the 300 ppm exposure, with approximately half of the proximal tubules lined by regenerating epithelium and an increased LI of tubule cells of 8-fold over control. In rats, mild centrilobular vacuolation was observed only in the livers of rats exposed to 300 ppm. The hepatocyte LI in rats were increased only at 100 and 300 ppm, 3- and 7-fold over control, respectively. In the kidneys of the male rats exposed to 300 ppm, about 25 to 50% of the proximal tubules were lined by regenerating epithelium. The LI for tubule cells in the cortex was increased at 30 ppm and above. In the nasal passages of rats, chloroform concentrations of 10 ppm and above induced histopathological changes that exhibited clear concentration-related severity. These lesions consisted of respiratory epithelial goblet cell hyperplasia and degeneration of Bowman's glands in olfactory mucosa with an associated osseous hyperplasia of the endo and ectoturbinates in the periphery of the ethmoid region. These nasal lesions were not observed in mice. Knowledge of the dose-dependent responses in rats and mice will be valuable in assessing the potential risks to humans posed by sinhaled chloroform and in setting exposure concentrations for longer-term studies.