The effects of subchronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus-pituitary-thyroid axis of male Fischer 344 rats.

Acrylamide (AA) is an important industrial chemical that is neurotoxic in rodents and humans and carcinogenic in rodents. The observation of cancer in endocrine-responsive tissues in Fischer 344 rats has prompted hypotheses of hormonal dysregulation, as opposed to DNA damage, as the mechanism for tumor induction by AA. The current investigation examines possible evidence for disruption of the hypothalamic-pituitary-thyroid axis from 14 days of repeated exposure of male Fischer 344 rats to doses of AA that range from one that is carcinogenic after lifetime exposure (2.5 mg/kg/d), an intermediate dose (10 mg/kg/d), and a high dose (50 mg/kg/d) that is neurotoxic for this exposure time. The endpoints selected include: serum levels of thyroid and pituitary hormones; target tissue expression of genes involved in hormone synthesis, release, and receptors; neurotransmitters in the CNS that affect hormone homeostasis; and histopathological evaluation of target tissues. These studies showed virtually no evidence for systematic alteration of the hypothalamic-pituitary-thyroid axis and do not support hormone dysregulation as a plausible mechanism for AA-induced thyroid cancer in the Fischer 344 rat. Specifically, there were no significant changes in: 1) mRNA levels in hypothalamus or pituitary for TRH, TSH, thyroid hormone receptor alpha and beta, as well 10 other hormones or releasing factors; 2) mRNA levels in thyroid for thyroglobulin, thyroid peroxidase, sodium iodide symporter, or type I deiodinases; 3) serum TSH or T3 levels (T4 was decreased at high dose only); 4) dopaminergic tone in the hypothalamus and pituitary or importantly 5) increased cell proliferation (Mki67 mRNA and Ki-67 protein levels were not increased) in thyroid or pituitary. These negative findings are consistent with a genotoxic mechanism of AA carcinogenicity based on metabolism to glycidamide and DNA adduct formation. Clarification of this mechanistic dichotomy may be useful in human cancer risk assessments for AA.

Analyses of bronchial bulky DNA adduct levels and CYP2C9, GSTP1 and NQO1 genotypes in a Hungarian study population with pulmonary diseases.

Carcinogen-DNA adducts may represent an intermediate end-point in the carcinogenic cascade and may reflect exposure to chemical carcinogens, as well as susceptibility and, ultimately, cancer risk. Interindividual variability in activity of enzymes involved in the metabolism of polycyclic aromatic hydrocarbons to mutagenic diol epoxides may predict adduct levels and, indirectly, lung cancer risk. Using 32P-postlabeling methods, the levels of bulky DNA adducts were determined in macroscopically normal bronchial tissues obtained from resected lobes of 143 Hungarian patients with lung malignancy and other pulmonary conditions. DNA from normal tissue was also evaluated for polymorphisms in cytochrome P450 2C9 (CYP2C9) at two sites, codons 144 (Arg/Cys) and 359 (Ile/Leu), for glutathione S-transferase P1 (GSTP1) at codon 105 and for NAD(P)H:quinone oxidoreductase (NQO1) at codon 187 (Pro/Ser). Using the Mann-Whitney U-test and analysis of variance, levels of adducts were evaluated in relation to variant genotypes, separately for smokers and non-smokers. As previously reported, bulky DNA adduct levels in smokers (n = 104) were estimated to be 54% higher than in non-smokers (n = 39) (8.6 +/- 4.2 versus 5.6 +/- 3.3 per 10(8) nucleotides, respectively, P < 0.01). Adduct levels were 16-29% higher in individuals with the homozygous Ile359/Ile359 CYP2C9 allele than in those heterozygous for the variant allele (Ile359/Leu359) [8.8 +/- 4.3 (n = 84) versus 7.6 +/- 3.5 (n = 20) for smokers and 5.8 +/- 3.5 (n = 32) versus 4.5 +/- 1.3 (n = 7) for non-smokers], although differences were not statistically significant. There were no clear differences in adduct levels in relation to genotypes of NQO1 or GSTP1. Although numbers of patients in this study are large in relation to many studies of carcinogen-DNA adducts, it is still possible that significant differences were not noted for polymorphisms in xenobiotic metabolizing enzymes due to relatively small numbers in stratified data.

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo.

Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study, we compared the extent of DNA adduct formation in female Sprague-Dawley rats treated by gavage with seven daily doses of 54 micromol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague-Dawley rats.

Exfoliated ductal epithelial cells in human breast milk: a source of target tissue DNA for molecular epidemiologic studies of breast cancer.

Studies of biomarkers of putative breast carcinogens, such as DNA adducts, have been limited by the difficulty in obtaining representative ductal epithelial cells (DECs) from breast tissue. In this feasibility study, we sought to ascertain if exfoliated DECs in breast milk could be a source of DNA for biomarker studies. Specimens (n = 38) were collected over 24 h from nursing women, and a questionnaire was administered. Cell pellets were isolated by repeated centrifugation and washing. Pellets were resuspended and incubated for 2 h, with glass adherence used to remove monocytes, resulting in an enrichment of DECs of >80%. Nonadherent cells were removed, washed, and homogenized for DNA isolation. Accurate DNA quantification was performed by 32P-postlabeling of normal nucleotides under conditions of excess ATP. Although there was wide variability in the amounts of DNA recovered, DNA yield was significantly associated with the number of weeks postpartum (P < 0.01), with optimal yield between 6 and 8 weeks after birth. There were no significant associations (P < 0.05) between the number of cells recovered and milk volume, method of collection, or the number of samples in a 24-h period per individual. This study demonstrates that breast milk can be used as a source of DECs for biomarker studies of gene-environment interaction and that sufficient DNA can be recovered to evaluate carcinogen-DNA adducts and to perform genotyping assays. Using this approach, exfoliated DECs may serve as a source of representative cells for studies of breast carcinogenesis and biomarkers of exposure, susceptibility, and effect.

Chemoprotection against the formation of colon DNA adducts from the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the rat.

The mutagenic heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), is a pyrolysis product in cooked foods that has been shown to be a rat colon carcinogen and has been implicated in the etiology of human colon cancer. In order to identify chemoprotection strategies that could be carried out in humans, a pilot study was conducted in which PhIP-DNA-adduct levels were quantified in the colons of male F344 rats that had been subjected to 16 different putative chemoprotection regimens, followed by a gavage of PhIP (50 mg/kg) and sacrifice 24 h later. The 16 treatments (Oltipraz, benzylisothiocyanate, diallyl sulfide, garlic powder, ethoxyquin, butylated hydroxyanisole, glutathione, indole-3-carbinol, alpha-angelicalactone, kahweol/cafestol palmitates, quercetin, green tea, black tea, tannic acid, amylase-resistant starch, and physical exercise) comprised sulfur-containing compounds, antioxidants, flavonoids, diterpenes, polyphenols, high dietary fiber, etc. The strongest inhibition of PhIP-DNA adduct formation in the colon was observed upon pretreatment with black tea, benzylisothiocyanate, and a mixture (1:1) of kahweol:cafestol palmitates, which resulted in 67, 66, and 54% decreases in colon PhIP-DNA adduct levels, as compared with controls. Preliminary studies on their mechanism of action indicated that only kahweol:cafestol caused a substantial induction of glutathione S-transferase isozymes (GSTs) that are thought to be important in the detoxification of PhIP. Notably, this induction occurred in the liver rather than in the colon.

Actions and interactions of nickel and magnesium on the transformation response of transformed cells in culture.

Nickel (Ni) and magnesium (Mg) exert separate and interacting effects on cells: Ni is toxic while Mg enhances the transformation response of transformed cells and protects from heavy metal-induced toxicity. Transformed rat liver epithelial cells were used in the soft agar (SA) assay to measure the effect of Ni and/or Mg on the expression of anchorage independence. Cells were exposed to +/- Ni and +/- Mg in a single passage of growth medium (GM) prior to assay in SA. The cells were then treated with +/- Ni and +/- Mg in the SA resulting in a 4 x 4 treatment matrix yielding 16 Ni/Mg combinations. Nickel was expected to decrease the transformation frequency (TF) and did so in 6 of the 16 cases. Magnesium was expected to enhance the TF independently of Ni; Mg increased TF values in 7 of 16 cases. The Ni-Mg interaction occurred in 11 of 16 cases. In general, Mg and Ni effects were observed more in GM than is SA. It is not evident from this study why the Ni, Mg, and Ni-Mg effects are not observed universally, but it is evident that metal-metal interactions are not simply defined or analyzed in biological systems. A refined factorial design may be useful in further separating such interactions. From the point of view of the implementation of the SA assay, in which test substances are typically dose previous to the implementation of putting the exposed cells in SA, it is clear that assay results can be markedly altered by the presence of the test compound in the soft agar.