Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism, cytochrome P-450 enzymes, and estrogen receptor alpha expression.

The objective of this study was to examine effects of estrogenic agents of varying potencies (genistein, p-nonylphenol, and ethinyl estradiol) on hepatic testosterone metabolism, cytochrome P-450 (CYP450) enzymes, and ERalpha expression. These endpoints were examined as potential biomarkers of, and contributors to, endocrine disruptive activity. Exposure occurred during critical developmental periods, from gestational day 7 through weaning via the mothers' diet. Thereafter, rats were exposed via their diet to the compounds until puberty (postnatal day 50). Testosterone hydroxylase and 5alpha-reductase activities, CYP2C and CYP3A levels were determined. In general, the compounds were more active in male rats than female rats. The only effect observed in female rats was at the 250 ppm genistein dose, in which an approximately 40% increase in 5alpha-reductase activity was observed. In male rats, genistein treatment had mixed effects on testosterone metabolism. The 1250 ppm dose decreased both CYP2C and CYP3A protein levels. Nonylphenol had the most profound effects on testosterone metabolism and CYP450 expression in male rats, with effects occurring at doses as low as 25 ppm. An increase in 5alpha-reductase activity and a decrease in the formation of 16alpha-OH-, 2alpha-OH-testosterone metabolites, CYP2C and CYP3A protein were observed. EE2 decreased the formation of several testosterone metabolites and CYP2C protein. All compounds had some effect on hepatic ERalpha expression, although a consistent effect was not observed. This study demonstrates that the test compounds can influence hepatic testosterone hydroxylase activity and CYP450 expression, as well as ERalpha expression, although these activities cannot be directly related to estrogenic activity.

Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A dose range-finding study was conducted as a prelude to a multigeneration bioassay to assess potential toxicities associated with genistein consumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625, or 1250 ppm to pregnant dams starting on Gestation day 7 and continuing throughout pregnancy. Dietary exposure of the dams continued through lactation, and pups were maintained on the same dosed feed as their mother after weaning until sacrifice at Postnatal day 50. Body weight and feed consumption of the treated dams prior to parturition showed a decreasing trend with a significant reduction at the highest dose. Litter birth weight was depressed in the 1250 ppm dose group, and pups of both sexes in that dose group had significantly decreased body weights relative to controls at the time of sacrifice. The most pronounced organ weight effects in the pups were decreased ventral prostate weight in males at the 1250 ppm dose and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at 250 to 1250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at 25 ppm and above. Abnormal cellular maturation in the vagina was observed at 625 and 1250 ppm, and abnormal ovarian antral follicles were observed at 1250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1250 ppm. There was a deficit of sperm in the epididymis at 625 and 1250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these doses. Both sexes showed an increase in the incidence and/or severity of renal tubal mineralization at doses of 250 ppm and above. Dietary genistein thus produced effects in multiple estrogen-sensitive tissues in males and females that are generally consistent with its estrogenic activity. These effects occurred within exposure ranges achievable in humans.

Polycystic kidney disease induced in F(1) Sprague-Dawley rats fed para-nonylphenol in a soy-free, casein-containing diet.

para-Nonylphenol (NP; CAS #84852-15-3), an alkylphenol with a 9-carbon olefin side chain, is widely used in the manufacture of nonionic surfactants, lubricant additives, polymer stabilizers, and antioxidants. Due to its wide commercial use and putative endocrine activity in humans and wildlife, the NTP elected to assess its effects on reproduction in multigenerational studies. To avoid known estrogenic activity of phytoestrogens in soy and alfalfa, a soy- and alfalfa-free, casein-containing diet was used in a range-finding study to determine the doses of NP to be tested further. NP was administered to Sprague-Dawley rats in the diet at 0, 5, 25, 200, 500, 1000, or 2000 ppm to F(0) dams beginning on gestation-day 7. The F(1) pups were weaned at postnatal day (PND) 21, and their exposure via diet was continued at the same dose level as their respective dams. Pup weights from birth through weaning were not significantly different from controls in any dose group, but the average weight of both sexes was significantly less compared to controls, beginning with the PND 28 weighing. The F(1) rats were sacrificed on PND 50 (n = 15, 3 pups of each sex from 5 litters for all dose groups). Terminal body weights of males and females in the 2000-ppm dose group were 74% and 85% of controls, respectively. Severe polycystic kidney disease (PKD) was present in 100% of the 2000 ppm-exposed male and female rats. At 1000 ppm, 67% of males and 53% of females had mild to moderate PKD versus none of either sex in the control and lower-dose groups. The no-adverse-effect level (NOAEL) for PKD was determined to be 500 ppm. Previous studies with comparable duration and route of exposure, but using soy-containing diets, reported either no or only mild PKD at 2000 ppm NP. We conclude that the renal toxicity of NP is highly dependent on the diet on which the animals are maintained. The potential interaction of diet and test compounds on nonreproductive as well as reproductive endpoints should be considered when contemplating the use of special diets formulated to minimize exogenous "hormone" content for the study of the effects of putative endocrine disruptive chemicals.

DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo.

The presence of relatively high levels of prostaglandin H synthase (PHS) in the dog urinary bladder and its ability to mediate the activation of carcinogenic arylamines to DNA-bound products in vitro suggests the involvement of this enzyme in arylamine-induced bladder carcinogenesis. Since the PHS-dependent metabolism of 2-naphthylamine (2-NA) had been shown to yield both ring- and N-oxidation products in vitro, we compared the reactivity of 3H-labeled N-hydroxy-2-naphthylamine (N-OH-2-NA), 2-nitrosonaphthalene, and 2-amino-1-naphthol (2-AN) toward DNA and protein. In the PHS-incubation system, all three derivatives bound at high levels to protein, but only N-OH-2-NA and 2-AN bound appreciably to DNA. Though ring-oxidation has usually been considered a detoxification pathway, the covalent binding of [3H]2-AN to DNA was found to occur readily under aerobic conditions and was enhanced at acidic pH. At pH 5 in air, the reactivity of [3H]2-AN with nucleic acids and protein was in the order: serum albumin greater than tRNA greater than poly G greater than poly C greater than DNA greater than poly A greater than rRNA greater than poly U. Enzymatic hydrolysis of DNA reacted with [3H]2-AN and subsequent analysis by h.p.l.c. indicated the presence of several carcinogen-nucleoside adducts. The major product was characterized as N4-(deoxyguanosin-N2-yl)-2-amino-1,4-naphthoquinoneimine; and two minor products were tentatively identified as N4-(deoxyadenosin-N6-yl)-2-amino-1,4-naphthoquinoneimine and a deoxyguanosin-N2-yl adduct of a naphthoquinoneimine dimer. These adducts accounted for approximately 60% of the total DNA binding obtained by incubation of [3H]2-NA with PHS in vitro and for approximately 20% of the [3H]2-NA bound to dog urothelial DNA in vivo. The remaining adducts were identical to those previously reported as products of the reaction of N-OH-2-NA with DNA. These results suggest that a minor proportion of the DNA adducts found in vivo may be formed by PHS-activation of 2-NA in the target tissue. Furthermore, the reactivity of 2-AN with cellular nucleophiles, presumably through formation of 2-imino-1-naphthoquinone or a protonated 4-naphthocarbenium ion, indicates that ring-oxidation products of arylamines and of other carcinogenic aryl compounds should be evaluated as proximate carcinogenic metabolites.

Metabolic oxidation of carcinogenic arylamines by rat, dog, and human hepatic microsomes and by purified flavin-containing and cytochrome P-450 monooxygenases.

Hepatic N-oxidation and aryl ring oxidation are generally regarded as critical activation and detoxification pathways for arylamine carcinogenesis. In this study, we examined the in vitro hepatic metabolism of the carcinogens, 2-aminofluorene (2-AF) and 2-naphthylamine (2-NA), and the suspected carcinogen, 1-naphthylamine (1-NA), using high-pressure liquid chromatography. Hepatic microsomes from rats, dogs, and humans were shown to catalyze the N-oxidation of 2-AF and of 2-NA, but not of 1-NA; and the rates of 2-AF N-oxidation were 2- to 3-fold greater than the rates of 2-NA N-oxidation. In each species, rates of 1-hydroxylation of 2-NA and 2-hydroxylation of 1-NA were comparable and were 2- to 5-fold greater than 6-hydroxylation of 2-NA or 5- and 7-hydroxylation of 2-AF. Purified rat hepatic monooxygenases, cytochromes P-450UT-A, P-450UT-H, P-450PB-B, P-450PB-D, P-450BNF-B, and P-450ISF/BNF-G but not P-450PB-C or P-450PB/PCN-E, catalyzed several ring oxidations as well as the N-oxidation of 2-AF. Cytochromes P-450PB-B, P-450BNF-B, and P-450ISF/BNF-G were most active; however, only cytochrome P-450ISF/BNF-G, the isosafrole-induced isozyme, catalyzed the N-oxidation of 2-NA. The purified porcine hepatic flavin-containing monooxygenase, which was known to carry out the N-oxidation of 2-AF, was found to catalyze only ring oxidation of 1-NA and 2-NA. No activity for 1-NA N-oxidation was found with any of the purified enzymes. These data support the hypothesis that 1-NA is probably not carcinogenic. Furthermore, carcinogenic arylamines appear to be metabolized similarly in humans and experimental animals and perhaps selectively by a specific form of hepatic cytochrome P-450. Enzyme mechanisms accounting for the observed product distributions were evaluated by Hückel molecular orbital calculations on neutral, free radical, and cation intermediates. A reaction pathway is proposed that involves two consecutive one-electron oxidations to form a paired substrate cation-enzyme hydroxyl anion intermediate that collapses to ring and N-hydroxy products.

Hepatic N-oxidation, acetyl-transfer and DNA-binding of the acetylated metabolites of the carcinogen, benzidine.

The metabolic N-oxidation, N-acetylation and N-deacetylation of the carcinogen benzidine (BZ) and its N-acetylated metabolites were examined in vitro with rat and mouse liver subcellular fractions. N-Oxidation of N-acetylbenzidine (ABZ) and N,N'-diacetylbenzidine (DABZ) was found to occur with NADPH-, NADH-fortified microsomes, although total oxidation at both nitrogens of ABZ was substantially faster than the N-oxidation of DABZ (four times for the mouse and 48 times for the rat). In both species, N-oxidation of ABZ to the arylhydroxylamine, N'-hydroxy-N-acetylbenzidine (N'-OH-ABZ), was somewhat faster than the formation of the arylhydroxamic acid, N-hydroxy-N-acetylbenzidine (N-OH-ABZ). N-Acetylation of BZ and ABZ by liver cytosol was quite efficient for both species (0.7-2.9 nmol/min/mg cytosolic protein), and these rates were found to be 3-10 times faster than their corresponding rates of N-oxidation. N-Deacetylation of ABZ and DABZ by mouse liver microsomes occurred at a rate that was comparable with N-acetylation; while N-deacetylation by rat liver microsomes was relatively slow, only 1-2% of the rate of N-acetylation. In the case of N-hydroxylated derivatives, N-OH-ABZ and N'-OH-ABZ, hepatic cytosolic N-acetylation by both rats and mice to form N-OH-DABZ was quite rapid (0.5-1.9 nmol/min/mg cytosol protein). Hepatic microsomal deacetylation of N-OH-DABZ also occurred with both species and was 2-4 times the rate of N-acetylation. These studies indicate that a significant concentration of potentially electrophilic monoacetylated N-oxidized metabolites may accumulate within the liver cell, and that they may serve as intermediates in the synthesis of the highly toxic metabolite, N-OH-DABZ. A major metabolic pathway for the formation of N-OH-DABZ is proposed as: BZ----ABZ----N'-OH-ABZ----N-OH-DABZ. The activation of N-OH-DABZ by cytosolic N,O-acyltransferase and N'-OH-ABZ by cytosolic sulfotransferase and O-acetyltransferase (acetyl CoA-dependent binding to DNA) were also examined. N-OH-DABZ N,O-acyltransferase and N'-OH-ABZ O-acetyltransferase were found to be significant pathways for rat and mouse liver, respectively. In addition, the DNA adduct formed from N-OH-DABZ in the presence of partially-purified rat hepatic N,O-acyltransferase was shown to be N'-(deoxyguanosin-8-yl)-N-acetylbenzidine, which is identical to that formed in rat liver in vivo and in the direct reaction of N'-OH-ABZ with DNA in vitro under acidic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of DNA adducts in vivo in rat liver and intestinal epithelium after administration of the carcinogen 3,2'-dimethyl-4-aminobiphenyl and its hydroxamic acid.

Administration of the 3H-labeled colon carcinogen, 3,2'-dimethyl-4-aminobiphenyl (DMABP) and its hydroxamic acid derivative, N-hydroxy-N-acetyl-DMABP, to male F344 rats resulted in high levels of covalent binding to hepatic and intestinal DNA, RNA and protein. For both compounds, binding to hepatic macromolecules was 2-4 times higher than in the intestine. High pressure liquid chromatographic analysis of the enzymatically hydrolyzed DNA from liver and intestinal epithelium indicated the presence of two carcinogen-DNA adducts: 5-(deoxyguanosin-N2-yl)-DMABP (15%), N-(deoxyguanosin-8-yl)-DMABP (50%), and a decomposition product of the latter (15%). N-acetylated adducts were not detected. When measured after 7 days, all adducts in the intestinal DNA had decreased by 70%, while only a 29% decrease had occurred in the hepatic DNA. To determine if the loss of DMABP products was a consequence of cell turnover or repair, rats were treated with [3H]thymidine and DMABP, and the specific activity of hepatic liver and intestinal DNA was measured. Between 1 and 7 days only a slight decrease in [3H]thymidine content occurred in hepatic DNA as compared with a 95% reduction in intestinal DNA. Thus, the higher rate of DNA synthesis in the intestine versus that in the liver may serve to promote fixation of the initiating lesion and account for the preferential induction of intestinal cancer by DMABP. Furthermore, comparison of these data with metabolic activation pathways reported earlier strongly suggest that N-hydroxy-DMABP is the proximate carcinogenic metabolite of both DMABP and N-hydroxy-N-acetyl-DMABP.

Characterization of the purine ring-opened 7-methylguanine and its persistence in rat bladder epithelial DNA after treatment with the carcinogen N-methylnitrosourea.

Purine ring-opened 7-methylguanine, prepared in vitro by alkaline treatment of 7-methylguanosine or of methylated calf thymus DNA, was extensively characterized by chromatographic and spectral techniques as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine. This modified base chromatographed as an early-eluting peak on an ion-exchange column but separated into two interconvertible components after reversed-phase or porous-resin h.p.l.c. The two components were analyzed by thermal desorption mass spectrometry and 500 MHz 1H-n.m.r. spectroscopy. Their mass spectra were identical (M+ at m/z 183) and their n.m.r. spectra each exhibited the same two sets of resonances whose relative intensities were solvent-dependent. Analysis by h.p.l.c. showed interconversion of the two components and kinetic studies demonstrated that this reaction was a reversible first-order process. At equilibrium, k1 = k2 = 0.334 h-1 and delta G = 22.9 kcal/mol. These data indicated that the ring-opened 7-methylguanine exists as cis/trans isomers with restricted rotation about the amide bond. Treatment of rats with an intraurethral initiating dose of the carcinogen N-methylnitrosourea resulted in a high level of bladder epithelial DNA modification with 7-methylguanine, O6-methylguanine, and methyl phosphotriesters as major adducts at 2 h after instillation. Purine ring-opened 7-methylguanine, chromatographically identical to the in vitro products, was initially a minor adduct. However, it was the only persistent modification in the bladder epithelial DNA and eventually accounted for 72% of the total carcinogen binding after 21 days. A tumor-promoting regimen, involving dietary sodium saccharin, did not alter the repair or persistence of any of the methylated adducts. These data demonstrate that purine ring-opened 7-methylguanine, previously reported to exist in liver DNA after N,N-dimethylnitrosamine or 1,2-dimethylhydrazine treatment, is present in a carcinogen-target tissue and is considerably more persistent than O6-methylguanine or other DNA methylation products. The possible role of this adduct as a promutagenic lesion initiating urinary bladder carcinogenesis is discussed.

Formation and persistence of DNA adducts from the carcinogen N-hydroxy-2-acetylaminofluorene in rat mammary gland in vivo.

The rat mammary carcinogen, N-hydroxy-2-acetylaminofluorene (N-hydroxy-2-AAF), has been proposed to be metabolically activated by mammary cytosolic N,O-acetyltransferase to a DNA binding species. To test this hypothesis, adult female Sprague-Dawley derived CD rats were treated, i.p., with 4.0 mg/kg [ring-3H]N-hydroxy-2-AAF. After 4 h, 1, 3, 14, and 28 days, the animals were killed, the mammary epithelium DNA was isolated and the carcinogen-deoxyribonucleoside adducts present were analyzed by high pressure liquid chromatography. At each time, only one adduct was detected and it was chromatographically identical to N-(deoxyguanosin-8-yl)-2-aminofluorene. The level of the adduct was maximal at 4 h (1.5 adducts/10(6) nucleotides) and then decreased, following first order kinetics with a t1/2 of 14.2 days. The detection of a single non-acetylated aminofluorene adduct is consistent with N,O-acyltransferase being involved in the metabolic activation of N-hydroxy-2-AAF in the rat mammary gland.

Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine.

A major and previously undetected carcinogen-DNA adduct was found in the livers of rats given N,N-dimethylnitrosamine or 1,2-dimethylhydrazine. This adduct, which accounted for 55% of the total methyl residues in DNA at 72 hours after carcinogen treatment, was chromatographically identical to a synthetic purine ring-opened derivative of 7-methylguanine and could be released from the isolated hepatic DNA by a specific E. coli glycosylase. The synthetic ring-opened adduct was characterized by mass and NMR spectroscopy as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine and appears to exist in two rotameric forms.

Rat mammary gland carcinogenesis after local injection of N-hydroxy-N-acyl-2-aminofluorenes: relationship to metabolic activation.

A single local injection of 2.5 mumol of N-hydroxy-N-formyl-2-aminofluorene (N-hydroxy-FAF), N-hydroxy-N-acetyl-2-aminofluorene (N-hydroxy-AAF), or N-hydroxy-N-pro-pionyl-2-aminofluorene )N-hydroxy-PAF) to each of the six left mammary glands of female Sprague-Dawley derived CD rats gave a mammary tumor incidence, after 12 months, of 53% for the N-acetyl (42% adenocarcinoma, 11% fibroadenoma), 41% for the N-formyl (8% adenocarcinoma, 11% sarcoma, 22% fibroadenoma), and 33% for the N-propionyl (11% adenocarcinoma, 22% fibroadenoma) derivatives of N-hydroxy-N-2-aminofluorene, Latent periods for malignant tumor appearance (adenocarcinoma or sarcoma) was 210 days, 148 days, and 177 days, respectively, with no malignant tumors occurring in the vehicle-treated animals. In contrast, latent periods for benign tumor appearance (fibroadenoma) was 263 days for control animals, 289 days for the N-hydroxy-AAF, 324 days for the N-hydroxy-FAF, and 317 days for the N-hydroxy-PAF animals. When N-acetyl-2-aminofluorene (AAF) was applied as above there was only an 8% mammary tumor incidence (4% adenocarcinoma, 4% fibroadenoma) with a latent period of 207 days for malignant tumor (adenocarcinoma) and 221 days for benign tumor (fibroadenoma) appearance. Arylhydroxamic acid N, O-acyltransferase activity has been demonstrated in the mammary glands of male, and lactating and non-lactating female Sprague-Dawley derived CD rats by means of nucleic acid binding assay. Mammary gland cytosol catalyzed tRNA adduct formation to a greater extent with N-hydroxy-FAF. AAF was not activated by this enzyme. Ammonium sulfate fractionation demonstrated the presence of two enzymes, one specific for N-hydroxy-FAF (70-80% fraction), the other specific for N-hydroxy-AAF and N-hydroxy-PAF (40-70% fraction). Moreover, gel filtration chromatography of mammary gland cytosol demonstrated the presence of two enzymes of differing acyl specificity. Mammary gland microsomes catalyzed the formation of tRNA adducts, but only with the N-hydroxy-FAF derivative. Assays that tested the mutagenic potential of the arylhydroxamic acids in Salmonella typhimurium TA-1538 with either mammary gland cytosol or microsomes demonstrated the order of mutagenicity to be N-hydroxy-FAF greater than N-hydroxy-AAF greater than N-hydroxy-PAF. A similar order of mutagenicity was demonstrated without an external metabolic activation system. These data demonstrate that the presence of two distinct enzymes in the rat mammary gland that activate arylhydroxamic acids.

A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography.

Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O2-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O2-alkylcytosines, 3-, O6-, and 7- alkylguanines and O2-, 3-, and O4-alkylthymines. N6-Alkyladenines, 1-alkylguanines and N2-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [14C]methyl methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.