Toxicology and carcinogenesis studies of a nondecolorized [corrected] whole leaf extract of Aloe barbadensis Miller (Aloe vera) in F344/N rats and B6C3F1 mice (drinking water study).

BACKGROUND: Extracts from the leaves of the Aloe vera plant (Aloe barbadensis Miller) have long been used as herbal remedies and are also now promoted as a dietary supplement, in liquid tonics, powders or tablets, as a laxative and to prevent a variety of illnesses. We studied the effects of Aloe vera extract on rats and mice to identify potential toxic or cancer-related hazards. METHODS: We gave solutions of nondecolorized extracts of Aloe vera leaves in the drinking water to groups of rats and mice for 2 years. Groups of 48 rats received solutions containing 0.5%, 1% or 1.5% of Aloe vera extract in the drinking water, and groups of mice received solutions containing 1%, 2%, or 3% of Aloe vera extract. Similar groups of animals were given plain drinking water and served as the control groups. At the end of the study tissues from more than 40 sites were examined for every animal. RESULTS: In all groups of rats and mice receiving the Aloe vera extract, the rates of hyperplasia in the large intestine were markedly increased compared to the control animals. There were also increases in hyperplasia in the small intestine in rats receiving the Aloe vera extract, increases in hyperplasia of the stomach in male and female rats and female mice receiving the Aloe vera extract, and increases in hyperplasia of the mesenteric lymph nodes in male and female rats and male mice receiving the Aloe vera extract. In addition, cancers of the large intestine occurred in male and female rats given the Aloe vera extract, though none had been seen in the control groups of rats for this and other studies at this laboratory. CONCLUSIONS: We conclude that nondecolorized Aloe vera caused cancers of the large intestine in male and female rats and also caused hyperplasia of the large intestine, small intestine, stomach, and lymph nodes in male and female rats. Aloe vera extract also caused hyperplasia of the large intestine in male and female mice and hyperplasia of the mesenteric lymph node in male mice and hyperplasia of the stomach in female mice.

Toxicological evaluation of brominated vegetable oil in Sprague Dawley rats.

Brominated vegetable oil (BVO) has been approved by the US Food and Drug Administration on an interim basis as a food additive. Past studies have raised concerns about potential toxicities from consuming BVO. To investigate further these toxicities, we conducted a 90-day dietary exposure study in Sprague Dawley rats and analyzed tissue distribution of the main metabolites. Six-week-old male and female rats were fed diets containing 0 (control), 0.002%, 0.02%, 0.1%, or 0.5% BVO by weight. Statistically significant increases were observed in the serum bromide in the high-dose group of both sexes and in the incidence of thyroid follicular cell hypertrophy in the two highest dose groups of males and the high-dose group of females. An increase in serum TSH was observed in the high-dose group for both sexes, as well as a decrease in serum T4 in the high-dose males. A clear dose-response was observed in di- and tetra-bromostearic acid levels in the heart, liver, and inguinal fat. These data expand upon previous observations in rats and pigs that oral exposure to BVO is associated with increased tissue levels of inorganic and organic bromine, and that the thyroid is a potential target organ of toxicity.

Oxidative stress related DNA adducts in the liver of female rats fed with sunflower-, rapeseed-, olive- or coconut oil supplemented diets.

Both animal and epidemiological studies support an effect of fatty acid composition in the diet on cancer development, in particular on colon cancer. We investigated the modulating effect of supplementation of the diet of female F344 rats with sunflower-, rapeseed-, olive-, or coconut oil on the formation of the promutagenic, exocyclic DNA adducts in the liver, an organ where major metabolism of fatty acids takes place. 1,N(6)-ethenodeoxyadenosine (etheno-dA), 3,N(4)-ethenodeoxycytidine (etheno-dC) and 1,N(2)-propandodeoxyguanosine from 4-hydroxy-2-nonenal (HNE-dGp) were determined as markers for DNA-damage derived from lipid peroxidation products and markers for oxidative stress. 8-Oxo-deoxyguanosine (8-Oxo-dG) was also measured as direct oxidative stress marker. The body weight of the rats was not influenced by the four diets containing the different vegetable oils during the 4-week feeding period. Highest adduct levels of etheno-dC (430 +/- 181 adducts/10(9) parent bases), HNE-dGp (617 +/- 96 adducts/10(9) parent bases) and 8-Oxo-dG (37,400 +/- 12,200 adducts/10(9) parent bases) were seen in rats on sunflower oil diet (highest linoleic acid content). Highest adducts levels of etheno-dA (133 +/- 113 adducts/10(9) parent bases) were found in coconut oil diet (lowest content of linoleic acid). Weakly positive correlations between linoleic acid content in the four diet groups were only observed for levels of HNE-dGp and 8-Oxo-dG. Neither the diet based on olive oil (which contains mainly oleic acid) nor the diet based on rapeseed oil (containing alpha-linolenic acid) exerted any significant protective effect against oxidative DNA damage. Our results indicate that a high linoleic acid diet may contribute to oxidative stress in the liver of female rats leading to a marginal increase in oxidative DNA-damage.

Circadian variation in the induction of intestinal tumors by N-methyl-N-nitrosourea in male C57BL/6N mice.

Male C57BL/6N mice were administered a single ip injection of 30 mg of N-methyl-N-nitrosourea (MNU)/kg of body weight. Additional groups were treated similarly every 3 hours for the next 24 hours. Adenocarcinomas of the small intestine were the major treatment-related tumors, with the total incidence being 38% at 250 days after injection. There was a significant circadian variation for tumor induction; the maximum number of intestinal tumors (approximately equal to 55%) tended to occur when the MNU was administered during the middle of the light period (6:00 to 18:00), while the tumor incidence was at a minimum (approximately equal to 10%) when the MNU was given in the middle of the dark phase (18:00 to 6:00). These data are discussed in relation to DNA synthesis and repair and MNU-induced cellular toxicity.

Acyltransferase-mediated binding of N-hydroxyarylamides to nucleic acids.

N-Hydroxyarylamides are metabolically activated to nucleic acid-binding species by the action of N,O-acyltransferase (AT). The substrate specificity of these enzymes in rat, guinea pig, monkey, baboon, pig, and human liver has been examined by measuring the AT-mediated nucleic acid binding of the N-formyl, N-acetyl, and N-propionyl derivatives of N-hydroxy-2-aminofluorene. Human and pig enzymes catalyzed binding in the order formyl greater than acetyl greater than propionyl, while for the other species the order was acetyl greater than propionyl greater than formyl. Ammonium sulfate fractionation of the cytosols suggested that the baboon and rat have at least two different AT's: one with a higher specificity for the formyl derivative; the other with a marked preference for acetyl and propionyl compounds. Only one form, with a high formyl group specificity, was detected from human liver. The identity of the in vitro AT-mediated DNA adducts from rat, baboon, and human liver was established. In each instance, one adduct accounted for greater than 75% of the bound 2-aminofluorene (AF) residues. This product had a high-pressure liquid chromatography retention time and pH-dependent partition characteristics identical to those of an adduct synthesized by an acid-dependent (pH 4.6) reaction of N-hydroxy-2-aminofluorene with calf thymus DNA. This synthetic adduct has been identified as N-(deoxyguanosin-8-yl)-2-aminofluorene by nuclear magnetic resonance, mass, and ultraviolet light spectroscopy. Moreover, it was identical to the product obtained from the alkaline (pH 12) hydrolysis of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene. Since an arylaminated (i.e., aminofluorene) residue(s) is the major product found in rat liver DNA following administration of N-hydroxy-N-acetyl-2-aminofluorene, these data suggest that AT may play a major role in the formation of this DNA-carcinogen adduct.

Persistence of DNA adducts in rat liver and kidney after multiple doses of the carcinogen N-hydroxy-2-acetylaminofluorene.

Male and female Sprague-Dawley rats were treated by gastric intubation either 1, 2, 3, or 4 times at biweekly intervals with 10-mg/kg doses of the hepatocarcinogen of N-[ring-3H]-hydroxy-2-acetylaminofluorene. Then either 1 or 14 days following the last treatment, the concentrations of 2-aminofluorene and 2-acetylaminofluorene adducts in liver and kidney DNA were established. 2-Acetylaminofluorene adducts were found in male rat liver DNA only. The C-8-acetylated adduct, N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, was detected only on the day following treatment at a concentration between 1.0 and 2.4 pmol/mg DNA. A second acetylated adduct, 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, was found at both 1 and 14 days after treatment and, as a result, increased in concentration with repeated doses, from 0.2 pmol/mg DNA after one dose to 2.8 pmol/mg DNA after four treatments. The major adduct detected in male rat liver and the only adduct found in female rat liver and in kidney from either sex was N-(deoxyguanosin-8-yl)-2-aminofluorene. This adduct was slowly lost from the DNA and therefore increased in concentration with repetitive treatments as follows: male liver, 4.0 to 9.4 pmol/mg DNA; female liver, 11.4 to 30.6 pmol/mg DNA; male kidney, 1.1 to 1.8 pmol/mg DNA; and female kidney, 1.8 to 17.7 pmol/mg DNA. These data indicate that certain DNA adducts can accumulate in both target and non-target tissues and therefore suggest that persistence of DNA adducts per se is not sufficient for tumor induction.

DNA adduct formation and T-lymphocyte mutation induction in F344 rats implanted with tumorigenic doses of 1,6-dinitropyrene.

Diesel emissions are known to induce tumors in experimental animals and are suspected of being carcinogenic in humans. Of the compounds associated with diesel exhaust, 1,6-dinitropyrene is a particularly potent mutagen and carcinogen. In these experiments, we have investigated the use of DNA adducts and T-lymphocyte mutations of 1,6-dinitropyrene as biomarkers for exposure to diesel emissions. 1,6-Dinitropyrene (0-150 micrograms) was applied directly to the lungs of male F344 rats according to a protocol known to induce lung tumors. In target (lung) and surrogate (liver, WBC, and spleen lymphocytes) tissues, one major DNA adduct, N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene, was detected by HPLC and/or 32P-postlabeling analyses. The levels of this adduct reached a maximum 1-7 days following treatment and decreased to 13-50% of the peak values by 28 days after dosing. In the lung, a 2-fold increase in dose resulted in a 2-fold increase in DNA binding up to the 30-micrograms dose; in the liver the same relationship was observed up to 10 micrograms 1,6-dinitropyrene. At higher doses, the extent of adduct formation still increased, but the rate was much lower than that occurring at lower doses. A limiting dilution clonal assay was used to measure mutation induction at the hypoxanthine-guanine phosphoribosyltranferase locus in spleen T lymphocytes. Following treatment, the mutant frequency increased until 21 weeks, remained constant until week 40, and then began to decrease. Mutant induction was dose related, with the increase in mutant frequency being significant at doses > or = 1 microgram 1,6-dinitropyrene. These data indicate that 1,6-dinitropyrene, a constituent of diesel emissions, is metabolically activated by nitroreduction to give DNA adducts in target and surrogate tissues. They further suggest that T-lymphocyte mutations may be a more sensitive and longer-lived biomarker than DNA adducts for assessing previous exposures to nitropolycyclic aromatic hydrocarbons.

Formation of DNA adducts in vitro and in Salmonella typhimurium upon metabolic reduction of the environmental mutagen 1-nitropyrene.

The polycyclic nitroaromatic hydrocarbon 1-nitropyrene is an environmental pollutant, a potent bacterial mutagen, and a carcinogen. Xanthine oxidase, a mammalian nitroreductase, catalyzed the in vitro metabolic activation of this compound to DNA-bound adducts. Maximum adduct formation occurred at pH 5.5 to 6.0 and was increased by the addition of catalase to the incubation medium. DNA binding from 1-nitropyrene was inhibited by hydrogen peroxide, L-ascorbate, and glutathione. Enzymatic hydrolysis of the modified DNA and subsequent analysis by high-pressure liquid chromatography indicated the presence of one major and two minor adducts. The major adduct was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy as N-(deoxyguanosin-8-yl)-1-aminopyrene. The minor adducts appear to be decomposition products of the major adduct. When Salmonella typhimurium TA1538 was incubated with 1-nitropyrene, a strong correlation was found between the extent of DNA binding and the frequency of induced histidine reversions. Analysis of the bacterial DNA indicated one major adduct which had chromatographic properties and pKaS identical to those of N-(deoxyguanosin-8-yl)-1-aminopyrene. These data indicate that N-hydroxy-1-aminopyrene is probably the mutagenic and DNA-binding species formed during the metabolic reduction of 1-nitropyrene.

Vaginal epithelial DNA damage and expression of preneoplastic markers in mice during chronic dosing with tumorigenic levels of 3'-azido-2',3'-dideoxythymidine.

3'-Azido-2',3'-dideoxythymidine (AZT, Retrovir, zidovudine), a nucleoside analogue currently used in the therapy of acquired immunodeficiency syndrome, induces papillomas and carcinomas in vaginal epithelium of mice as a result of lifetime drug administration. In this study, female CD-1 mice were administered AZT at doses of 180, 360, and 720 micrograms/ml of drinking water for 28 days to determine whether AZT became incorporated into vaginal DNA and whether this was associated with preneoplastic changes within the target tissue. In addition, bone marrow, a target for AZT-induced cytotoxicity in mice and humans, was examined for chromosomal aberrations. A positive correlation was observed between dose level of AZT, proliferation of cells in the basal layer of vaginal epithelium, and incorporation of AZT into vaginal DNA. Incorporation of AZT into vaginal DNA was originally detected by radioimmunoassay and confirmed by immunohistochemistry. An aberrant pattern for alpha 6 integrin distribution, similar to the pattern described in skin papillomas with high risk for malignant conversion, also increased with dose in mice given AZT. Chromosomal aberrations in bone marrow increased more than 4-fold in AZT-exposed animals. The genotoxicity demonstrated by incorporation of AZT into vaginal DNA and proliferation of vaginal epithelium may play an essential part in the ability of AZT to induce abnormal differentiation in vaginal epithelium and vaginal tumorigenesis in mice.

Local carcinogenicity, rates of absorption, extent and persistence of macromolecular binding, and acute histopathological effects of N-hydroxy-1-naphthylamine and N-hydroxy-2-naphthylamine.

The N-hydroxy derivatives of 1- and 2-naphthylamine (NA) are directly carcinogenic at sites of application. In this study, the carcinogenicity of these two compounds at s.c. injection sites was compared with their relative rates of absorption, with the extent and persistence of their binding to protein, RNA, and DNA in the skin-subcutis, and with acute histopathological changes observed after local application. Male Sprague-Dawley rats were given injections of the N-hydroxy derivatives in the right rear leg at 16 mumol/dose. When administered twice weekly for 12 weeks, N-hydroxy-1-NA caused a 100% incidence (30 of 30) of poorly differentiated sarcomas at the injection site. N-Hydroxy-2-NA administered in a similar manner resulted in a low yield of tumors (7%; 2 of 30). Injection of N-hydroxy-1-NA once weekly for 12 weeks or twice weekly for 6 weeks also induced a high incidence of sarcomas (93 to 97%), but the time to tumor formation was significantly longer (p less than 0.0001) than in animals treated twice weekly for 12 weeks. The tumors were classified as malignant fibrous histiocytomas. Possible antagonistic or synergistic effects between the two compounds were also investigated. A sequential 6-week treatment with each of the N-hydroxy derivatives did not alter the expected tumor yields. However, alternating injections over 12 weeks caused a significant lengthening in the time to tumor formation (p less than 0.05). N-Hydroxy-1-NA bound covalently to protein, RNA, and DNA to a much greater extent than did N-hydroxy-2-NA. Protein binding with both derivatives decreased by 80 to 90% by 7 days after treatment. RNA binding in N-hydroxy-1-NA-treated rats markedly decreased, while N-hydroxy-2-NA-bound residues diminished only slightly. During this period, the extent of DNA binding with both derivatives remained fairly constant. When N-hydroxy-2-NA was injected 3 days after N-hydroxy-1-NA, there was a marked reduction in the apparent levels of N-hydroxy-1-NA bound to RNA and DNA. The greater tumorigenicity of N-hydroxy-1-NA versus N-hydroxy-2-NA correlated with its greater extent of macromolecular binding. Examination of acute histopathological changes occurring after single injections of N-hydroxy-1-NA and/or N-hydroxy-2-NA indicated that both compounds caused extensive necrosis in tissues at the injection site.(ABSTRACT TRUNCATED AT 400 WORDS)

Covalent binding of benzidine and N-acetylbenzidine to DNA at the C-8 atom of deoxyguanosine in vivo and in vitro.

Benzidine, a human urinary bladder carcinogen, induces hepatic tumors in mice and rats. In this study, [3H]benzidine was administered in drinking water to mice for 1 week, and the covalent binding of the carcinogen to hepatic DNA was then determined. A single carcinogen:DNA adduct was detected which decreased in concentration by approximately 50% at 1 day after treatment and then remained at a nearly constant level for at least 7 days. Injection of radiolabeled benzidine or N-acetyl-benzidine into rats also resulted in a single carcinogen:DNA adduct that was chromatographically identical to that obtained in mouse liver. While administration of benzidine and N-acetylbenzidine resulted in high levels of the adduct in rat hepatic DNA, injection of N,N'-[ring-14C]diacetylbenzidine did not give detectable binding (less than 0.3 residue/mg DNA). The same carcinogen:DNA adduct found in rat and mouse liver was prepared synthetically by: (a) hydrolysis of calf thymus DNA reacted with N-hydroxy-N'-acetylbenzidine at pH 5; and (b) reaction of N-acetoxy-N,N'-diacetylbenzidine with deoxyguanosine and subsequent selective deacetylation of the product with methanolic ammonia. The in vitro and in vivo products were found to have identical high-pressure liquid chromatography retention times and to exhibit similar pH-dependent solvent partitioning characteristics. Mass and nuclear magnetic resonance spectral data on the synthetic products established the structure of the hepatic adduct as N-(deoxyguanosin-8-yl)-N'-acetylbenzidine. The structural isomer, N-(deoxyguanosin-8-yl)-N-acetylbenzidine, was synthesized by treatment of N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine (the intermediate in b) with carboxylesterase and was shown to be chromatographically distinct from the in vivo adduct. Similarly, the nonacetylated derivative, N-(deoxyguanosin-8-yl) benzidine, was synthesized by carboxylesterase treatment of N-(deoxyguanosin-8-yl)-N'-acetylbenzidine and was shown not to occur in rat and mouse liver DNA. These data indicate that the metabolic activation of benzidine to an ultimate carcinogen in rats and mice does not involve N-hydroxybenzidine or sulfotransferase-catalyzed activation of N-hydroxy-N,N'-diacetylbenzidine. The remaining pathways for metabolic conversion of benzidine to an ultimate carcinogenic species are discussed in relation to liver and urinary bladder carcinogenesis.

Metabolic activation of N-hydroxy-N,N'-diacetylbenzidine by hepatic sulfotransferase.

N-Hydroxy-N,N'-diacetylbenzidine (N-HO-DABZ) has been shown to be an in vitro metabolite of benzidine in several rodent species and may represent the proximate form of the carcinogen. Like other arylhydroxamic acids, N-HO-DABZ may be converted to an ultimate carcinogenic electrophile by metabolic O-sulfonation in hepatic cytosol. To investigate this possibility, liver cytosols from rats, mice, and hamsters were assayed for their ability to catalyze the 3'-phosphoadenosine 5'-phosphosulfate-dependent metabolism of N-HO-DABZ and the formation of an adduct with methionine. For comparative purposes, sulfotransferase activity for N-hydroxy-2-acetylaminofluorene (N-HO-AF) was also measured. In the rat, N-HO-DABZ and N-HO-AAF were metabolized at rates of 2.5 and 4.3 nmol of arylhydroxamic acid lost per in per mg of protein, respectively. In the mouse, these rates were 0.5 nmol for N-HO-DABZ and less than 0.05 nmol for N-HO-AAF. Sulfotransferase activity for these substrates in hamster liver cytosol could not be detected (less than 0.05 nmol/min/mg). The inclusion of methionine in sulfotransferase incubation mixtures and subsequent heating resulted in the formation of methylmercapto arylamides from both N-HO-DABZ and N-HO-AAF. From 20 to 40% of the N-HO-DABZ metabolized was trapped and recovered as an adduct, while 80 to 100% of the N-HO-AAF metabolized was similarly obtained. A methylmercapto-N,N'-diacetylbenzidine derivative was also obtained by reaction of N-acetoxy-N,N'-diacetylbenzidine with methionine. Its identity to the adduct formed in the sulfotransferase incubation mixture was established by high-pressure liquid chromatography, ultraviolet light, and mass spectroscopic analyses. By comparing the 13C nuclear magnetic resonance spectra of the synthetic methylmercapto derivative with several model compounds and using chemical shift additivity relationships, the adduct was identified as 3-methylmercapto-N,N'-diacetylbenzidine. Since the yield of the product from N-acetoxy-N,N'-diacetylbenzidine and methionine did not vary appreciably with pH (4 to 8), a reaction mechanism involving an electrophilic carbocation at position 3 is proposed. These studies demonstrate that N-HO-DABZ can be esterified to an electrophilic reactant by hepatic sulfotransferases in the rat and the mouse and suggest the involvement of this metabolite in the hepatocarcinogenicity of benzidine.

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.

Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats.

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (∼6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-2'-deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 10(8) nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo.

Development of a novel (32)P-postlabeling method for the analysis of 3'-azido-3'-deoxythymidine.

3'-Azido-3'-deoxythymidine (AZT) was the first anti-retroviral nucleoside analog to be used in the treatment and prevention of acquired immunodeficiency syndrome (AIDS). A novel (32)P-postlabeling assay, based upon thymidine kinase (TK) instead of the conventional T(4) polynucleotide kinase, has been developed for the detection of the levels of AZT incorporated into DNA. After enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates, AZT was isolated by ethyl acetate extraction. The ethyl acetate was evaporated and the AZT was postlabeled by 5'-phosphorylation with [gamma-(32)P]ATP and TK. AZT was detected at a level of 51.5+/-6.3 (mean+/-SD; n=4) AZT molecules/10(5) nucleotides following in vitro incorporation of the drug into high-molecular-weight rat-liver DNA. The (32)P-postlabeling method was further validated by the detection of AZT in lung and liver DNA from neonatal mice treated with AZT. The levels of AZT in lung and liver DNA were proportional to the dose, with the levels in lung DNA being two-fold higher than those for liver DNA. The limit of detection for the assay was 8 AZT molecules/10(7) nucleotides using 10 microg of DNA.

Ab initio study on the molecular structure of trans-1,2-dihydroxy-1,2-dihydro-8-fluoronaphthalene.

The molecular geometries of two conformations (diequatorial and diaxial) of trans-1,2-dihydroxy-1,2-dihydro-8-fluoronaphthalene have been refined the ab initio gradient method at the 4-21G level to determine the effect of fluoro substitution on the conformational and structural properties of naphthalene dihydrodiols. As with trans-1,2-dihydroxy-1,2-dihydronaphthalene, the conformation with diequatorial hydroxyl groups is the most stable. The structural differences for the fluorinated and unfluorinated naphthalene dihydrodiols are discussed and the possible consequences of the structural and conformational trends on the metabolism of dihydrodiols to dihydrodiol epoxides are considered.

Formation of DNA adducts and oxidative DNA damage in rats treated with 1,6-dinitropyrene.

In vitro metabolism studies have indicated that the tumorigenic environmental pollutant 1,6-dinitropyrene has the potential to bind covalently to DNA and to induce oxidative DNA damage. We have determined if 1,6-dinitropyrene treatment will cause both types of DNA damage in vivo. Female Sprague-Dawley rats were given a single intraperitoneal injection of 1,6-dinitropyrene, and covalent DNA adduct formation, as indicated by the presence of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene, and oxidative DNA damage, as indicated by increases in 5-hydroxymethyl-2'-deoxyuridine and 8-hydroxy-2'-deoxyguanosine, were assessed at 3, 12, 24 and 48 h after dosing. 32P-postlabeling analyses of DNA isolated from liver, mammary gland, bladder and nucleated blood cells indicated the formation of N-(deoxy-guanosin-8-yl)-1-amino-6-nitropyrene, with the levels being highest in the bladder. 5-hydroxymethyl-2'-deoxyuridine was detected in DNA from each of these tissues, and the levels of this oxidized nucleoside were higher in the mammary glands and livers of 1,6-dinitropyrene-treated rats. 1,6-Dinitropyrene dosing did not affect the levels of 8-hydroxy-2'-deoxyguanosine in these two tissues. These results indicate that exposure to 1,6-dinitropyrene can result in increased levels of 5-hydroxymethyl-2'-deoxyuridine in addition to covalent DNA adduct formation.

Interactive effects of methyl-deficiency and dietary restriction on liver cell proliferation and telomerase activity in Fischer 344 rats pretreated with aflatoxin B(1).

The effects of methyl-deficiency and dietary restriction (DR) on hepatic cell proliferation and telomerase activity was studied in male Fischer 344 rats pretreated with aflatoxin B(1) (AFB(1)). Five-week-old rats were gavaged 5 days per week for 3 weeks with AFB(1) (25 microg/rat per day) or solvent (100 microl 75% dimethylsulfoxide). Rats were then divided into four groups. Two groups were fed a methyl-sufficient (MS) diet either ab libitum (AL) or with DR. The other two groups were fed a methyl-deficient (MD) diet either AL or with DR. At 15, 20, and 32 weeks of age, hepatic cell proliferation, telomerase activity, and the number of glutathione S-transferase-P positive (GST-P(+)) foci were determined. DR reduced hepatic cell proliferation, while the MD diet and AFB(1) pretreatment increased cell proliferation. Telomerase activity was decreased by DR and increased by the MD diet and AFB(1) pretreatment. The same trend was observed with GST-P(+) foci: in AFB(1)-pretreated rats, methyl deficiency increased the number of foci, while DR decreased the number. These results are consistent with a role of telomerase in hepatocarcinogenesis.

Hprt lymphocyte mutant frequency in relation to DNA adduct formation in rats fed the hepatocarcinogen 2-acetylaminofluorene.

The lymphocyte hypoxanthine-guanine phosphoribosyltransferase (Hprt) assay is frequently used as a biomarker for the exposure of both humans and laboratory animals to potentially carcinogenic agents. To obtain information concerning the sensitivity of the rat Hprt lymphocyte assay toward aromatic amine carcinogens, male F344 rats were fed 0.02% 2-acetylaminofluorene (2-AAF) for 1 month and then returned to control diet for 2 months. At 4, 27, 48, 62, and 90 days after the initiation of 2-AAF-feeding, the frequency of mutants in the Hprt gene was determined. In addition, DNA was isolated from liver nuclei, spleen lymphocytes, bone marrow, and thymus, and DNA adducts were analyzed by 32P-postlabeling. 2-AAF feeding resulted in a significant induction of 6-thioguanine-resistant T-lymphocytes and the mutant frequency continued to increase after the 2-AAF feeding was stopped. The same major DNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, was detected in liver, spleen lymphocytes, bone marrow, and thymus. DNA adduct levels were greatest in the tumor target tissue (liver) but occurred in all T-lymphocyte compartments, being highest in spleen lymphocytes. The DNA adduct levels were highest at the end of the 1-month 2-AAF feeding period and decreased rapidly in all tissues. The data indicate that the Hprt lymphocyte mutagenesis assay detects arylamine carcinogens, but with relatively low sensitivity.

Induction of rat hepatic cytochromes P-450 by environmental nitropolycyclic aromatic hydrocarbons.

Nitrated polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that result from various incomplete combustion processes. We have examined the activity of hepatic microsomal enzymes in rats pretreated with a series of environmentally occurring nitrated PAHs including: 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 6-nitrochrysene, 7-nitrobenz[a]anthracene, 3-nitrofluoranthene, and 1-, 3-, and 6-nitrobenzo[a]pyrene. None of the compounds increased the cytochrome P-450 content more than 2-fold. 1,8-Dinitropyrene, 6-nitrochrysene, and 1- and 3-nitrobenzo[a]pyrene significantly increased arylhydrocarbon hydroxylase activity 2- to 8-fold higher than solvent-treated controls. The induction of 7-ethoxycoumarin O-deethylase activity paralleled that found with arylhydrocarbon hydroxylase. The maximum induction of aminopyrine N-demethylase was only 1.5-fold, and none of the nitrated PAHs caused significant increases in epoxide hydrase or NADPH-cytochrome c reductase. 1-Nitropyrene reductase activity was induced by each of the compounds with the exception of 6-nitrobenzo[a]pyrene. The greatest increase was caused by 1-nitrobenzo[a]pyrene followed by 1,3-dinitropyrene, 3-nitrobenzo[a]pyrene and 6-nitrochrysene. These data suggest that nitrated PAHs may potentiate the effects of subsequent exposures to various chemical carcinogens.