Dioxin in soil: bioavailability after ingestion by rats and guinea pigs.

Soil environmentally contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was given by gavage to guinea pigs and rats. The development of a characteristic clinicopathologic syndrome in guinea pigs, the induction of aryl hydrocarbon hydroxylase in rats, and the presence of TCDD in the livers of both species show that TCDD in soil exhibits high biological availability after ingestion.

Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer.

BACKGROUND: Numerous studies have associated bladder cancer with exposure to carcinogens present in tobacco smoke and other environmental or occupational exposures. Approximately 50% of all humans inherit two deleted copies of the GSTM1 gene which encodes for the carcinogen-detoxification enzyme glutathione S-transferase M1. Recent findings suggest that the GSTM1 gene may modulate the internal dose of environmental carcinogens and thereby affect the risk of developing bladder cancer. PURPOSE: We investigated whether the absence of the GSTM1 gene affects bladder cancer risk and whether there are racial differences in GSTM1 genotype frequency. METHODS: Using a polymerase chain reaction (PCR)-based method, we examined the frequency of the homozygous deleted genotype (GSTM1 0/0) in 229 patients with transitional cell carcinoma of the bladder and 211 control subjects who were enrolled from the Urology Clinics at Duke University Medical Center and the University of North Carolina Hospitals. Control subjects were urology clinic patients who primarily presented with benign prostatic hypertrophy or impotence, who had no history of any cancer other than nonmelanoma skin cancer, and who were frequency matched to case patients on race, sex, and age (10-year age intervals). In order to explore racial differences in GSTM1 gene frequency, genotype was also determined in a community-based sample of 466 paid, healthy, unrelated volunteers from Durham and Chapel Hill, N.C. The presence or absence of the GSTM1 gene locus was determined by using a differential PCR, a semiquantitative technique in which multiple genes are coamplified. RESULTS: Overall, the GSTM1 0/0 genotype conferred a 70% increased risk of bladder cancer (odds ratio [OR] = 1.7; 95% confidence interval [CI] = 1.2-2.5; P = .004). Absence of the GSTM1 gene encoding the glutathione S-transferase M1 enzyme significantly increased risk to persons with exposure to the carcinogens in tobacco smoke (OR = 1.8; 95% CI = 1.2-3.0; P = .01) but poses little increased risk to persons without such exposure. Persons with smoking exposure of more than 50 pack-years who had the GSTM1 0/0 genotype had a sixfold greater risk relative to persons in the lowest risk group (i.e., nonsmokers who were GSTM1 +/+ or +/0). In the pooled clinic control and community sample groups (677 individuals), the GSTM1 0/0 genotype occurred less frequently among Blacks (35%) than among Whites (49%, P < .001). CONCLUSIONS: These findings support a protective role for the GSTM1 gene in bladder cancer. From these findings, it is estimated that 25% of all bladder cancer may be attributable to the at-risk GSTM1 0/0 genotype.

alpha-Naphthoflavone metabolized by 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced rat liver microsomes: a potent clastogen in Chinese hamster ovary cells.

alpha-Naphthoflavone (ANF) is a widely used inhibitor of P-450-mediated metabolism. Previously, we have demonstrated that in vitro addition of ANF to human lymphocytes produced significantly greater numbers of sister chromatid exchanges (SCEs) in samples from smokers compared to nonsmokers. In order to study the mechanism of this differential induction, we investigated the clastogenic activity of ANF as a consequence of metabolism by induced and uninduced rat liver microsomes. Exponentially growing Chinese hamster ovary cells were treated with ANF for 2 h in the presence or absence of microsomes, followed by incubation for 12 (chromosome aberrations) or 24 h (SCEs). ANF induced concentration (4 to 40 microM)-dependent increases in SCEs and chromosome aberrations when coincubated with 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced microsomes. At the lower concentrations of ANF, chromatid damage was most predominant, whereas at the higher concentrations, a high percentage of cells was killed. The surviving cells exhibited shattered chromosomes and multiple damage in the form of chromatid exchanges and breaks. ANF was not clastogenic nor did it induce SCEs in Chinese hamster ovary cells when incubated with microsomes from control rats or phenobarbital-treated rats. Moreover, NADPH was required for the clastogenic actions of ANF in the presence of 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced microsomes. Analysis of the ANF metabolites by high-pressure liquid chromatography revealed that 2,3,7,8-tetrachlorodibenzo(p)dioxin-induced microsomes metabolized ANF to a much greater extent than control or phenobarbital-induced microsomes. Our results suggest that the clastogenic activity of ANF in Chinese hamster ovary cells is mediated by the cytochrome P-450 monooxygenase system.

Continued development of hepatic gamma-glutamyltranspeptidase-positive foci upon withdrawal of 17 alpha-ethynylestradiol in diethylnitrosamine-initiated rats.

Adult ovariectomized Sprague-Dawley rats were administered a single initiating dose of 200 mg diethylnitrosamine (DEN)/kg, i.p. 17 alpha-ethynylestradiol (EE2) was then chronically administered to the rats by means of s.c. Silastic implants at an estimated dose of 90 micrograms EE2/kg/day. Hepatic gamma-glutamyltranspeptidase-positive foci were evaluated after 20-60 (+20,+30,...,+60) weeks of chronic EE2 treatment and after 20 or 30 weeks of EE2 followed by 20 or 30 weeks with no EE2 [(+20,-20), (+30,-20), (+30,-30)] to determine the effects of withdrawal of the promoting agent on the persistence or reversibility of these focal lesions. Our results show that gamma-glutamyltranspeptidase-positive foci are no longer dependent on exogenous EE2 administration for their continued growth in initiated animals given EE2 chronically for 20 weeks. In DEN-initiated, EE2-promoted animals the number of foci per cc (Nv) seen at 20 weeks increased over the next 20 weeks in the absence of further EE2 treatment, but was not statistically different than Nv for continued EE2 treatment. The proportion of total liver volume occupied by foci (Vv) was 0.0054 (+30), 0.0191 (+30,-20), and 0.0135 (+50). The (+30,-20) Vv was significantly different than that for (+30) (P less than 0.01). Hepatocellular adenomas and carcinomas were detected in DEN-initiated and EE2- promoted animals as early as 30 weeks. Hepatic tumor incidence continued to increase after withdrawal of EE2 in initiated animals which had received only 30 weeks of promotion. Within the framework of our studies, the promoting effects of EE2 do not appear to be reversible by withdrawal of EE2 after 20 weeks of treatment. It may be that events or factors which were estrogen dependent in the early stages of promotion are now constitutive.

Changes in estrogen receptor, DNA ploidy, and estrogen metabolism in rat hepatocytes during a two-stage model for hepatocarcinogenesis using 17 alpha-ethinylestradiol as the promoting agent.

17 alpha-Ethylestradiol (EE2) was administered chronically to diethylnitrosamine (DEN)-initiated (200/mg/kg, i.p.) adult ovariectomized Sprague-Dawley rats, by means of Silastic implants at an estimated dose of 90 micrograms/kg/day. Isolated hepatocytes from DEN/EE2-treated animals exhibited a 2- to 3-fold increase in nuclear estrogen receptor (ER) levels throughout the promotion period. Furthermore, approximately 30-40% of the receptor was occupied when quantified by an exchange assay. For all groups the ER had a sedimentation coefficient of approximately 8S for unoccupied ER and a binding affinity for 17 beta-estradiol of 0.25 nM. An ER of lower affinity for estradiol was present in animals initiated with DEN and/or promoted with EE2. The increase in hepatocyte ER was associated with a 5.2-fold increase in gamma-glutamyl transpeptidase and 2.5-fold decrease in glucose-6-phosphatase activity at 20 weeks. EE2 treatment caused a 50% increase in the maximal binding capacity (Bmax) of hepatic epidermal growth factor receptors, but the equilibrium binding constant (Kd) did not change. Modulation of mitotic activity of hepatocyte subpopulations by EE2 treatment was indicated by an increase in the proportion of diploid hepatocytes and an increase in the number of hepatocytes undergoing DNA synthesis. In general, effects on ER, epidermal growth factor receptor, gamma-glutamyl transpeptidase and glucose-6-phosphatase were greater in DEN/EE2-treated animals than in rats receiving only EE2. Modification of receptor pathways associated with hepatocyte growth control, ER and epidermal growth factor receptor, may be contributing factors in the clonal expansion of preneoplastic cells during EE2 promotion of hepatocarcinogenesis.

Ovarian tumors in rats induced by chronic 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multispecies reproductive toxicant, and it has been recently classified by IARC as a known human carcinogen. Here, we report that TCDD promotes the development of ovarian tumors in an initiation-promotion model in female Sprague Dawley rats. Rats were initiated with diethylnitrosamine (DEN) or vehicle at 70 days of age. Starting 2 or 18 weeks after initiation, rats were exposed biweekly to TCDD at a daily average dose of 125 ng/kg/day for 14, 30, or 60 weeks continuously or for 30 weeks plus withdrawal periods of 16 or 30 weeks. Fifteen of 76 (20%) rats initiated with DEN and promoted with TCDD for various lengths of time developed ovarian sex cord-stromal tumors of Sertoli cell type, whereas no ovarian tumors developed in 86 rats used as vehicle controls or that received DEN alone or TCDD alone. The highest tumor incidence occurred in 6 of 14 rats (43%) after 60 weeks of continuous TCDD after DEN initiation. One of six rats developed a tumor by 30 weeks of exposure. Because most effects of TCDD can be attributed to its activation of the aryl hydrocarbon receptor (AhR), the presence and localization of AhR was determined in the rat ovary and in the ovarian tumors by reverse transcription-PCR, immunohistochemistry, and in situ hybridization. AhR was localized to oocytes, granulosa and thecal cells of growing follicles, surface epithelial cells, and epithelial cells lining single tubules in ovaries from adult control Sprague Dawley rats. Neoplastic cells in the ovarian tumors were also positive for both AhR message and protein. These results indicate that the ability of TCDD to cause ovarian tumors is dependent on initiation, length of promotion, and age of the animal when exposed and evaluated. The tumor type induced by TCDD in this experimental system is the same histological subtype as that reported from an early study of youngsters exposed during an industrial accident in Seveso, Italy.

Relationships among benzo(a)pyrene metabolism, benzo(a)pyrene-diol-epoxide:DNA adduct formation, and sister chromatid exchanges in human lymphocytes from smokers and nonsmokers.

In the present study, benzo(a)pyrene (BP) metabolism, DNA adduct formation, ethoxyresorufin-O-deethylase activity, and sister chromatid exchange induction by BP were compared in human lymphocytes prepared from whole blood of smokers and nonsmokers following an in vitro incubation with BP. There was an approximate 7- to 10-fold variation in all parameters measured. To determine the source of this variation, participants were resampled, the assays were repeated, and all the data were analyzed to assess (a) smoking-related effects, (b) differences in multiple samples from the same individual, and (c) intraindividual, experimental, and interindividual variation. No smoking-related effects were observed except for baseline sister chromatid exchange frequency. The variation observed for BP-related DNA adducts and ethoxyresorufin-O-deethylase activity was primarily due to interindividual variation. For example, in vitro formation of DNA adducts did not change when samples were obtained at different times from the same individual and were not influenced significantly by culture conditions. No significant correlation existed between DNA adduct formation and BP metabolism [correlation coefficient (r) = 0.27] for either the total population or when segregated based on smoking status. Furthermore, no correlation was seen between DNA adducts and sister chromatid exchange induction by BP. Our studies have compared a number of commonly used lymphocyte markers and conclude that it is difficult to predict changes in one marker based on changes in another. However, in vitro formation, of PB-derived DNA adducts is consistent over time for individuals.

Effects of alpha-naphthoflavone on levels of sister chromatid exchanges in lymphocytes from active and passive cigarette smokers: dose-response relationships.

The frequency of sister chromatid exchanges (SCE) were determined in lymphocytes of nonsmokers, passive smokers, and active smokers in the presence and absence of alpha-naphthoflavone (ANF). Higher levels of SCEs were detected for all smoking groups after in vitro addition of ANF when compared with an assay without ANF. There was a highly statistically significant difference between heavy smokers and nonsmokers (9.25 versus 7.43 SCE/cell) for the assay without ANF and for the ANF assay (14.2 versus 8.8). When considering the numerical difference in SCEs between the assays with and without ANF (delta SCE), higher values were noted for moderate smokers (2.7) and heavy smokers (4.9) compared to nonsmokers (1.4). Significant dose-response relationships were found between the frequency of SCEs and factors related to smoking, such as duration and frequency of cigarette use, tar, nicotine, carbon monoxide content of brand, and urinary measures of nicotine metabolites (cotinine and thiocyanate). No elevation of SCEs in passive smokers was found when compared to nonsmokers using either assay. The mechanism for SCE enhancement by ANF is unclear, but may be related to metabolic activation of the ANF by the cytochrome P-450 system in lymphocytes. The dosimetry relationships between cigarette smoke exposure and SCE frequency indicate that culture of human lymphocytes via ANF may provide a sensitive tool to detect exposure to cigarette smoke.

Dose-response relationships for chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in a rat tumor promotion model: quantification and immunolocalization of CYP1A1 and CYP1A2 in the liver.

The mechanisms responsible for the braod spectrum of effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are not entirely clear but seem to involve an initial interaction with the Ah receptor. A major uncertainty in risk assessment for TCDD is the lack of adequate dose-response relationships following chronic exposure to TCDD. Induction of cytochrome P-450 enzymes (CYP1A1 and CYP1A2) is one of the most sensitive responses to TCDD and its structural analogues. We have used a two-stage model for hepatocarcinogenesis in female Sprague-Dawley rats to evaluate dose-response relationships for induction of CYP1A1 and CYP1A2 in diethylnitrosamine-initiated as well as in noninitiated rats. After initiation with a single dose of diethylnitrosamine, TCDD was administered biweekly by p.o. gavage at doses equivalent to 3.5, 10.7, 35.7, and 125 ng/kg/day for 30 weeks. CYP1A1 and CYP1A2 concentrations were quantified in hepatic microsomes by radioimmunoassay and localized in hepatic tissue slices by immunohistochemical techniques. Radioimmunoassay data revealed a maximum induction of 200-fold for CYP1A1 and 10-fold for CYP1A2 and there were no statistically significant differences between initiated and noninitiated rats. Induction at the lowest dose (3.5 ng/kg/day) was 20-fold for CYP1A1 and 3-fold for CYP1A2. Mathematical analysis indicates that the best fit of the induction data are inconsistent with a threshold for this response. There was a linear relationship between administered dose and TCDD liver concentration over the entire dose range of the study. This indicates that induction of CYP1A2 does not significantly alter the distribution of TCDD in our chronic dosing regimen. Immunolocalization of CYP1A1 and CYP1A2 revealed the same localization and induction pattern for both isozymes in the cytoplasm of hepatocytes. However, the hepatic distribution pattern was not uniform with the most intense staining observed around central veins. These studies help to clarify dose-response relationships for dioxin-mediated effects and demonstrate different sensitivity of hepatocytes to the effects of TCDD.

Ovarian hormones enhance 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated increases in cell proliferation and preneoplastic foci in a two-stage model for rat hepatocarcinogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent hepatocarcinogen in rodents. However, liver tumor incidence is increased by TCDD in female Sprague-Dawley rats but not male rats in chronic carcinogen bioassays. Our studies have investigated this finding by evaluating histological and biochemical parameters in a two-stage model for hepatocarcinogenesis in female Sprague-Dawley rats (intact and ovariectomized), using diethylnitrosamine (DEN) as the initiating agent and TCDD as the promoting agent. Increases in gamma-glutamyl transpeptidase-positive foci were greater in intact female rats than in ovariectomized (OVX) animals. For example, in intact rats receiving both DEN and TCDD, the percentage of liver occupied by gamma-glutamyl transpeptidase-positive foci was 0.37, compared to 0.08 in OVX rats. Values for intact or OVX rats receiving either DEN or TCDD only were 0.04 or less. Similar results were obtained when using placental glutathione S-transferase to detect hepatic preneoplastic lesions. Cell proliferation data, obtained using bromodeoxyuridine in osmotic minipumps, were consistent with preneoplastic foci data in that the hepatocyte labeling index was increased in DEN/TCDD intact rats but not in DEN/TCDD OVX rats. Analysis of data from individual animals revealed a strong correlation (P less than 0.01) between cell proliferation and placental glutathione S-transferase-positive foci/cm3 in liver. These findings did not reflect effects of ovariectomy on TCDD tissue distribution, since livers of OVX rats contained more TCDD than livers of intact rats, although both groups of rats received a dose of 1.4 micrograms TCDD/kg once every 2 weeks for 30 weeks. Hepatic cytochrome P-450d (IA2) was induced approximately 6-8-fold in all TCDD-treated groups, and the magnitude of induction was not influenced by ovariectomy. This cytochrome efficiently catalyzes metabolism of 17 beta-estradiol to catechol estrogens. Our data suggest that ovarian hormones (probably estrogen) play a significant role in the hepatocarcinogenic actions of TCDD.

Dioxin-responsive genes: examination of dose-response relationships using quantitative reverse transcriptase-polymerase chain reaction.

The purpose of the present experiments was to examine dose-response relationships for induction of hepatic mRNA following a single administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other "dioxin-responsive" genes including UDP-glucuronosyltransferase I, plasminogen activator inhibitor 2, and transforming growth factor alpha using a sensitive reverse transcriptase-polymerase chain reaction-based method. Sample-to-sample variability in amplification is a concern in using polymerase chain reaction to quantitate biological responses. However, in the present study recombinant RNA templates were synthesized to use as internal standards in both the reverse transcription and the polymerase chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive to TCDD treatment with increases observed at doses as low as 1 ng/kg body weight. The induction of CYP1A1 mRNA correlated highly (R2 > 0.90) with an increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated enzyme activity. However, induction of CYP1A1 mRNA levels was detected at lower TCDD doses than was ethoxyresorufin-o-deethylase activity, reflecting the greater sensitivity of the reverse transcription-polymerase chain reaction approach to detect transcriptional activation of the CYP1A1 gene. UDP-glucuronosyltransferase I mRNA was increased over control (5-fold) but required 1000-times higher TCDD doses (1 microgram/kg) to result in a significant increase than did CYP1A1. Plasminogen activator inhibitor 2 and transforming growth factor alpha mRNA, both previously shown to be induced by TCDD in human keratinocytes, were not increased in rat liver. Hence, these studies reaffirm that TCDD acts through classical receptor mechanisms with gene-to-gene differences in responsiveness. The reverse transcription-polymerase chain reaction method developed to measure mRNA for dioxin-responsive genes in rat liver will allow for measuring multigene and tissue responses to TCDD and other xenobiotics with high sensitivity, reproducibility, and adaptability and should increase our understanding of various dose-response relationships.

Hepatic and extrahepatic N-acetyltransferase. Perinatal development using a new radioassay.

We report a sensitive and rapid radioassay method for p-aminobenzoic acid N-acetyltransferase. The principle of this assay involves acetylation of p-aminobenzoic acid with [1-14C] labeled acetyl coenzyme A and direct extraction of enzymically formed radioactive p-acetamidobenzoic acid into nonaqueous scintillation fluid. Using this radiometric assay, hepatic and extrahepatic tissue distributions from rat and rabbit were studied. Rabbit blastocyst and endometrial N-acetyltransferase specific activities were equivalent to hepatic activities. Perinatal development studies in rats and rabbits revealed that fetal and neonatal animals are capable of N-acetylation. Rat liver developmental studies exhibited two peaks of activity with the first peak occurring in the late fetus followed by a second peak 3 days after birth. Rabbit fetal and neonatal enzyme activity increased to adult levels by the second week after birth in liver and gut, however, lung showed a different developmental pattern. These studies demonstrate that fetal extrahepatic tissues, like adult tissues, play an important role in N-acetylation.

Sexual dimorphism of the chromatographic profiles of I-compounds (endogenous deoxyribonucleic acid modifications) in rat liver.

DNA of all tissues studied thus far in untreated mammals contains as yet structurally unidentified, covalent modifications termed I (indigenous)-compounds, which are detectable by the 32P postlabeling assay for DNA adducts and increase with age. The purpose of this study was to determine the effects of sex, gonadectomy, and androgen administration on I-compound profiles and levels in order to gain insight into the factors involved in the biosynthesis of these DNA modifications. Liver DNA from various groups of 6-month-old Sprague-Dawley rats (untreated or gonadectomized males and females; animals with or without gonadectomy treated with testosterone propionate) was analyzed by a nuclease P1-enhanced version of the 32P postlabeling assay. Hepatic I-compound profiles of untreated animals exhibited pronounced sexual dimorphism. In addition to a number of I-compounds that differed quantitatively between sexes, 7 female-specific and 1 male-specific I-compounds were observed. In female rats, the total level amounted to 112 I-compounds in 10(9) DNA nucleotides and exceeded the level in males by 3-fold. Castration feminized and ovariectomy masculinized I-compound profiles and levels. Neonatal testosterone propionate failed to restore the male pattern of I-compounds lost by neonatal castration, so that an androgen-imprinting mechanism did not appear to be involved in the maintenance of the male I-compound phenotype and the suppression of the female pattern. Testosterone propionate administered to intact female animals lowered total I-compound levels significantly. The results indicate that estrogens play a dominant role in regulating sex-dependent formation of I-compounds in rat liver. The dependence of I-compound formation on both age and sex hormones suggests that the levels of these DNA modifications are developmentally controlled.

Effect of gonadectomy on the ontogeny of estrogen-binding components in rat liver cytosol.

These studies elucidate the ontogeny of two classes of estrogen-binding sites present in rat liver cytosol. The first class of sites is precipitated from whole cytosol fractions by ammonium sulfate (30% saturation) and exhibits characteristics of specific estrogen receptors. Detectable levels of receptors are attained during the third postnatal week. During days 30--40, receptors reach maximum concentration and remain relatively constant thereafter. The second class of sites, detected in whole cytosol fractions, possess a high binding capacity for estrogens and are present in similar amounts in male and female liver before day 34. However, between days 34--40 male levels increase dramatically while female levels remain constant. This sex difference is maintained throughout the duration of the study (160 days). Specific estrogen receptors from immature (26 days) and mature (70--80 days) rat liver have similar characteristics in terms of sedimentation properties in sucrose gradients, ligand binding specificity, and heat and pronase susceptibility. After prepuberal (19--21 days) gonadectomy, levels of receptor in subsequent adult animals of both sexes are increased approximately 40%. No alterations in receptor levels are seen after neonatal (day 1) castration of males. Prepuberal (day 19) gonadectomy does not alter the normal development of sexual differentiation of high capacity estrogen-binding sites. However, after neonatal (day 1) castration male rats, levels of those sites do not undergo sexual differentiation, and neonatally castrated adult males exhibit female levels of high capacity estrogen-binding site. These studies suggest that sexual differentiation of high capacity estrogen-binding sites may be programmed at birth by testicular androgens.

Imprinting of hepatic estrogen-binding proteins by neonatal androgens.

Previous studies discovered a second class of estrogen-binding proteins distinct from estrogen receptor which exhibited higher capacity, lower affinity (HCLA) binding properties. HCLA sites underwent postpubertal sex differentiation, such that adult male levels were at least 10-fold higher than adult female levels. Neonatal castration of male rats prevented the subsequent sex differentiation of HCLA binding sites; adult male rats that were castrated neonatally exhibited typically female concentrations of these binding sites. If male rats were castrated at 19 days of age or later, postpubertal sex differentiation of HCLA binding sites proceeded as observed for intact males. Administration of testosterone propionate (TP) to castrated (neonatally) males during a critical period (days 6-13) imprinted for the subsequent sex differentiation of HCLA sites, whereas TP administration at other times did not. The expression of these imprinted sites was not manifested until puberty, as neonatal manipulation or TP treatment had no effect on HCLA sites in immature rats. The imprinted effect on HCLA binding sites appeared to be permanent and irreversible. Treatment of castrate males with diethylstilbestrol (DES) or zearalenol (P-1496) during the critical period was incapable of restoring development of normal male levels of HCLA sites. Competitive binding studies using several steroid hormones revealed similar data for intact males and castrate (neonatal) male rats that had received TP during the critical period. These studies demonstrate an early imprinting period during which androgen exposure programs for postpubertal development of sex differentiation of HCLA binding sites.

Ethnic variation in the CYP2E1 gene: polymorphism analysis of 695 African-Americans, European-Americans and Taiwanese.

Human cytochrome P4502E1 (CYP2E1) is inducible by ethanol and is involved in metabolism of many known carcinogens including N-nitrosodimethylamine, butadiene, benzene, and carbon tetrachloride. A 50-fold variability in CYP2E1 enzyme activity in humans has been observed but it is unknown whether the basis for this variation is genetic or environmental. Recently, two restriction fragment length polymorphisms (RFLPs) within the CYP2E1 gene have been suggested as genetic markers of risk for cancer. The first was a Rsa I polymorphism in the 5' regulatory region that appeared to alter transcriptional activation of the gene and the second was a Dra I polymorphism located approximately 7000 bp downstream in an intron. Rare alleles at each of these loci have been associated with a reduced risk for lung cancer in Japanese and Swedish populations. We have used a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to determine the genotype frequency for each of these CYP2E1 RFLPs in 695 individuals of Taiwanese, African-American or European-American background. Genotype and allele frequencies for Taiwanese were significantly different from those of African-Americans and European-Americans at either Rsa I or Dra I sites (p < 0.0001). Allele frequencies for African-Americans and European-Americans were significantly different at the Rsa I site (p = 0.03). The rare alleles (c2 and C) occurred at frequencies of 0.28 and 0.24 in Taiwanese, 0.01 and 0.08 in African-Americans, and 0.04 and 0.11 in European-Americans. In addition, we describe three haplotypes common to all three population samples and a fourth haplotype that was only detected in the Taiwanese population sample. This fourth haplotype may have been caused by a recombination event between these markers.(ABSTRACT TRUNCATED AT 250 WORDS)

Association between CYP1A1 genotype, mRNA expression and enzymatic activity in humans.

Genetic susceptibility factors may play a role in determining adverse effects of exposure to environmental toxins. As a preliminary step to a molecular epidemiological study in a population exposed to 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), we investigated 20 healthy Caucasian volunteers with a set of putative susceptibility markers including a CYP1A1 Msp I restriction fragment length genetic polymorphism (RFLP), CYP1A1 mRNA expression, and ethoxyresorufin-O-deethylase (EROD) activity in cultured and mitogen-activated blood lymphocytes. Both basal (p = 0.008) and induced (p = 0.0001) EROD activity was significantly higher among persons with a mutation in one or both alleles of the CYP1A1 gene (variant CYP1A1 genotype). Induction in vitro by TCDD significantly increased EROD activity in both variant and wild-type CYP1A1 subjects; however, the absolute increase was greater in subjects with variant genotypes. An additive interaction between genotype and TCDD induction was suggested. Expression of CYP1A1 mRNA, both basal and induced, did not vary significantly across the genotypes.

Central projections of the ethmoidal nerve of the cat as determined by the horseradish peroxidase tracer technique.

The ethmoidal nerve innervates the nasal mucosa and constitutes the afferent limb of several upper airway protective reflexes. Protective reflexes, such as sneezing, coughing, and apnea, are those reflexes that either expel foreign substances from the respiratory tract or stop them from gaining access to the lungs. The afferents for nasal receptors are thought to be a part of the trigeminal system rather than olfactory in nature. The objective of this study was to localize the cell bodies of these ethmoidal afferents and to trace the central projections of these neurons. Horseradish peroxidase was applied to the ethmoidal nerve in 11 adult cats. Following a survival period of 48-72 hours, the animals were killed and the tissue was processed according to the tetramethylbenzidine method. Reaction product was localized in cell bodies within the trigeminal ganglion, concentrated caudal to the entrance of the ophthalmic trunk of the trigeminal nerve. Transganglionic projections to the spinal trigeminal nucleus were localized primarily in the subnucleus interpolaris and in layers I and II of the subnucleus caudalis. There was also reaction product in cell bodies within the mesencephalic trigeminal nucleus. These results are in keeping with projections of other ophthalmic division receptor afferents, such as the cornea and the supraorbital nerve.

Trigeminal nuclear complex of the ferret: anatomical and immunohistochemical studies.

In order to establish the ferret as an animal model for studies of trigeminal pain, we describe the cytoarchitecture and neurochemistry of the trigeminal nuclear complex in the ferret and compare them to those of the cat and rat. The complex was divided as previously described, but the ferret differed in the extent of the nuclear boundaries. The neuroanatomical istribution of substance P-, calcitonin gene-related peptide-, galanin-, enkephalin-, serotonin-, somatostatin-, neuropeptide Y-, and neurotensin-immunoreactivity was determined throughout the rostrocaudal extent of the complex. In subnucleus caudalis, substance P-, calcitonin gene-related peptide-, enkephalin-, serotonin-, somatostatin-, neuropeptide Y-, and galanin-immunoreactivity was densest in laminae I and II. In subnucleus interpolaris, immunoreactivity for all the above neurochemicals was most dense along the lateral border and the ventral third of the caudal part of the subnucleus. Enkephalin-immunoreactive cell bodies were present in subnucleus caudalis and interpolaris. In subnucleus oralis, labelling for substance P, calcitonin gene-related peptide, galanin, enkephalin, and serotonin was most prominent in the dorsomedial part of the subnucleus. Somatostatin-immunoreactive cell bodies were distributed throughout the spinal nucleus. Labelling of serotonin, substance P, calcitonin gene-related peptide, galanin, enkephalin, and somatostatin was present in the main sensory nucleus. The motor nucleus contained fibers immunoreactive for substance P, enkephalin, serotonin and neuropeptide Y, and cell bodies immunoreactive for calcitonin gene-related peptide. The majority of neurotensin-immunoreactivity was found at the level of subnucleus caudalis, where it was densest in the trigeminal extension of the lateral cervical nucleus. The distribution of peptides in this species throughout the spinal nucleus is consistent with the notion that all the subnuclei may be involved in the processing of nociceptive inputs.

Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) results in a broad spectrum of biological responses, including altered metabolism, disruption of normal hormone signaling pathways, reproductive and developmental effects, and cancer. Cytochrome P450 1B1 (CYP1B1) is a dioxin-inducible gene that is active in the formation of 4-hydroxyestradiol, a potentially genotoxic catechol estrogen. Therefore, the analysis of CYP1B1 in humans may be useful in establishing relationships between dioxin exposure and adverse health effects. In this study, we examined the expression of CYP1B1 in human peripheral blood lymphocytes of unexposed individuals using a quantitative reverse transcription-PCR method. Absolute CYP1B1 RNA levels varied more than 30-fold in uncultured mononuclear cells obtained from 10 individuals. In vitro treatment of mitogen-stimulated lymphocytes with TCDD for 1-5 days of culture resulted in a peak induction of CYP1B1 after 3 days. The induction of CYP1B1 RNA levels after 3 days of culture was dose-dependent, exhibited a maximum response above 10 nM TCDD, and varied greatly among different individuals. However, the half maximal dose required for this induction was similar between individuals and comparable to that observed in the MCF-7 and HepG2 human cell lines. These observations indicate that CYP1B1 exhibits variable constitutive expression and is inducible in vitro by TCDD in human lymphocytes and that the magnitude of induction varies within the population. These data define the suitability of CYP1B1 for use as a mechanistically based biomarker in ongoing molecular epidemiological studies of human populations exposed to dioxins and related chemicals that bind the aromatic hydrocarbon receptor.