Detection of smoking-related covalent DNA adducts in human placenta.

The presence of covalent DNA chemical addition products (adducts) in human term placentas was investigated by recently developed immunologic and 32P-postlabeling assays. DNA from placental specimens of smokers showed a small but not statistically significant increase in adduct levels when tested by antibodies to DNA modified with a benzo[a]pyrene dihydrodiol epoxide (BPDE-I), the ultimate carcinogenic derivative of benzo[a]pyrene. The postlabeling assay detected several modified nucleotides, one of which (adduct 1) strongly related to maternal smoking during pregnancy. This adduct was present in placental tissue from 16 of 17 smokers, but only 3 of 14 nonsmokers. Among smokers, levels of adduct 1 in general were only weakly related to questionnaire and biochemical measures of the intensity of smoking exposures, which suggests modulation by individual susceptibility factors. The adduct seemed to be derived from an aromatic carcinogen, but it may not result from several of the most intensely studied polycyclic aromatic hydrocarbons or aromatic amines in tobacco smoke. The data show the association of cigarette smoking with covalent damage to human DNA in vivo.

Quantitative associations between DNA damage in human placenta and maternal smoking and birth weight.

Specimens of human placental DNA were tested for chemical addition products (adducts) by recently developed 32P-postlabeling and immunologic assays, and results were compared with data concerning maternal exposures and birth weight. A total of 7 different adducts were detected in the 53 specimens of human placental tissue examined by the 32P-postlabeling assay. Three of these adducts were found almost exclusively in smokers. Among smokers there were positive dose-response relationships between levels of the smoking-related adducts and biochemical estimates of doses of maternal exposure to cigarette smoke during pregnancy. Levels of 1 adduct found only in smokers appeared to relate directly to amounts of caffeine consumption by the mother. In addition to these relationships with maternal exposures, levels of smoking-related adducts were inversely associated with the birth weight of offspring. Results from this study suggest that even at their current formative stage of development, assays for DNA adducts may help identify determinants of DNA damage to human tissues and improve our ability to demonstrate dose-response relationships for the effects of environmental exposures to potentially carcinogenic agents.

PIP: The use of P-postlabeling assay to find the determinants of DNA damage demonstrates an innovative approach for measuring toxic end points. This approach begins with estimates of biochemical or molecular damage to tissue and then figures in exposure or susceptibility factors that are associated with the damage. Using this method, researchers studied smoking-related DNA adducts (chemical addition products) and found 7 different DNA adducts in human placental tissue specimens. Maternal smoking during pregnancy was related to 3 of the adducts observed. Levels of adducts were associated with caffeine use, age, race and education of the mother. The intensity of the smoking was determined by three methods: a questionnaire; a biological approach that scored smoking exposure and combined data from questionnaires and assays and a molecular approach that was related to levels of DNA adducts. There was a clear association between birth weight and birth length and levels of adduct that demonstrated that cigarette smoke does cause low birth weight and birth length. Little correlation between levels of tar, nicotine or carbon monoxide and adduct levels was found. The information and study was at an inchoate stage; therefore, further comparison and interpretation are needed to assess the findings.

Covalent DNA damage in tissues of cigarette smokers as determined by 32P-postlabeling assay.

Covalent DNA addition products (adducts) formed by the reaction of chemical carcinogens or their metabolites with DNA are critically involved in the initiation of chemical carcinogenesis and may serve as molecular markers and dosimeters for environmental carcinogen exposures. Using a highly sensitive 32P-postlabeling assay for DNA adduct analysis, we studied DNA damage elicited by cigarette smoke in tissues of smokers. A multitude of characteristic smoking-induced, presumably aromatic DNA adducts were found to occur in a dose- and time-dependent manner in the lung, bronchus, and larynx of smokers with cancer of these organs and to decline only slowly after cessation of smoking. Low levels of adducts appeared to persist for up to 14 years in the lungs of exsmokers with high previous exposures. These results corroborate data of epidemiological studies showing that the lung cancer risk and mortality of smokers increase with the intensity and duration of smoking and decline only slowly after cessation of smoking. Tissue distribution studies in autopsy samples revealed the presence of smoking-associated DNA lesions also in the kidney, bladder, esophagus, heart, ascending aorta, and liver. The most extensive DNA damage was found in lung and heart, i.e., 1 aromatic adduct in about 10(7) DNA nucleotides. Our results suggest that cigarette smoking-induced DNA adduct formation is causally related to cancer in the target organs.

Specific lack of the hypermodified nucleoside, queuosine, in hepatoma mitochondrial aspartate transfer RNA and its possible biological significance.

Tumor nucleic acids have frequently been found to be deficient in methylated and other modified nucleotides. In particular, cytoplasmic transfer RNAs (tRNAs) from various neoplasms partially lack the hypermodified nucleoside queuosine, a modification specific for anticodons of histidine-, tyrosine-, asparagine-, and aspartic acid-accepting tRNAs. Using aspartate tRNA as an example, we show here that liver mitochondria contain tRNA fully modified with respect to queuosine, while the corresponding tRNA from mitochondria of Morris hepatoma 5123D completely lacks this constituent. The sequences of these tRNAs, which were determined by a highly sensitive 32P-postlabeling procedure entailing the direct identification of each position of the polynucleotide chains, were found to be (sequence in text) Lack of queuosine in the hepatoma mitochondrial tRNA may be due to the inavailability of queuine in the hepatoma mitochondria for incorporation into tRNA or to inhibition of the modifying enzyme, tRNA (guanine)-transglycosylase, in the tumor. Taking into account results of others indicating a possible involvement of the queuosine modification in differentiation of eukaryotic cells, we hypothesize that the queuosine defect may develop at an early stage of carcinogenesis (i.e., during the promotion phase) and be directly involved in abnormalities of mitochondria which have been observed frequently in transformed cells and tumors.

Inhibition of estrogen-induced renal carcinogenesis in male Syrian hamsters by tamoxifen without decrease in DNA adduct levels.

Estrogens have previously been shown to induce covalent DNA modifications specifically in the hamster kidney, the target organ of estrogen-inducible and -dependent renal carcinoma. The DNA adducts, formed by yet unknown mechanisms, have been postulated to mediate hormonal carcinogenesis in this animal model. In an attempt to study a possible involvement of estrogen receptor mechanisms in the formation of DNA adducts, 17 beta-estradiol and the antihormone tamoxifen were concomitantly administered as s.c. implants to male Syrian hamsters. 17 beta-Estradiol-treated and tamoxifen-treated animals served as positive and negative controls, respectively. The tumor incidence decreased from 100% in 17 beta-estradiol-treated controls to 25% in the group receiving tamoxifen in addition to hormone. Tamoxifen-treated animals did not develop kidney tumors and did not show any detectable DNA damage. DNA adduct levels were comparable in hamsters treated with 17 beta-estradiol and 17 beta-estradiol plus tamoxifen for 5 or 7 months. In hamsters inoculated with H-301 cells, which are derived from the estrogen-induced hamster renal carcinoma and are estrogen dependent for growth, tamoxifen decreased estrogen-dependent H-301 tumor growth. However, in cell culture, neither 17 beta-estradiol nor tamoxifen influenced H-301 cell division. It was concluded that tamoxifen inhibited the growth of estrogen-induced renal carcinoma but did not interfere with tumor initiation since it did not inhibit the formation of DNA adducts. Moreover, receptor mechanisms were most probably not involved in the induction of DNA modifications by estrogens.

Lack of a specific ribose methylation at guanosine 17 in Morris hepatoma 5123D tRNASer1IGA.

Tumor transfer RNA's (tRNA's) frequently exhibit alterations in column chromatographic profiles and in base compositions when compared to their normal counterparts. Because such alterations may be involved in the dedifferentiated state of cancer cells, it is of interest to determine their structural basis and functional significance. The recent development of highly sensitive postlabeling methods has now made possible sequence analysis of tRNA's from neoplastic tissues available only in limited amounts. We have determined the nucleotide sequence of Morris hepatoma serine tRNA (anticodon IGA) and compared it with its normal counterpart in rat liver. The tumor serine tRNA was found to lack the ribose methylation of guanosine in position 17 of the dihydrouridine loop present in the liver RNA. This result explains the column chromatographic shifts of Morris hepatoma 5123D seryl-tRNA isoacceptors, suggesting that all seryl-tRNA isoacceptors may lack this modification.

Localization of estrogen-induced DNA adducts and cytochrome P-450 activity at the site of renal carcinogenesis in the hamster kidney.

Renal carcinoma in male Syrian hamsters, induced by chronic administration of estradiol for 5-7 months, is known to arise in the cortex at the cortico-medullary junction. In this in vivo model for hormonal carcinogenesis, estrogen-induced covalent DNA adducts have previously been observed in whole kidney and have been postulated to be involved in tumor induction. In the present study, the intrarenal distribution of estrogen-induced DNA modification and estrogen metabolizing enzymes were investigated in male Syrian hamsters to ascertain a role of metabolism and adduct formation in estrogen-induced carcinogenesis. The highest estrogen-induced DNA adduct concentrations as measured by 32P-postlabeling analysis were found in the renal cortex of hamsters treated with estradiol for 7 months. Total adduct levels in medullary DNA were approximately one-half of those found in cortex. Cytochrome P-450 enzymes were detected only in microsomes of kidney cortex (approximately 0.8 +/- 0.6 nmol P-450/mg protein) but not medulla of untreated male Syrian hamsters. Prostaglandin endoperoxide synthase activity in kidney cortical microsomes was 1/5 of the activity found in medullary microsomes. Thus, microsomal cytochrome P-450 levels and estrogen-induced DNA adduct formation were highest in hamster kidney cortex, the origin of renal tumorigenesis. It is postulated that estrogen metabolism by cytochrome P-450 enzymes leading to covalent DNA modification plays a role in hormonal carcinogenesis in the hamster kidney.

Comparative 32P-analysis of cigarette smoke-induced DNA damage in human tissues and mouse skin.

Previous studies using a highly sensitive 32P-postlabeling assay for the analysis of carcinogen/mutagen-induced DNA damage have shown the presence of tobacco smoking-related DNA adducts in human placenta (Everson, R.B., Randerath, E., Santella, R.M., Cefalo, R.C., Avitts, T. A., and Randerath, K., Science (Wash. DC), 231: 54-57, 1986). The occurrence of such adducts in smokers' bronchus and larynx is reported here. Since the chemical nature of these adducts could not be characterized by direct methods due to the extremely low levels of individual adducts (less than 0.03 fmol per microgram DNA), we have sought an experimental animal model for studying the formation of tobacco-related DNA adducts. Because cigarette smoke condensate is known to initiate tumors in mouse skin, ICR mice were treated topically with cigarette tar equivalent to 1.5, 3, 6, and 9 cigarettes for 0.4, 3, 5, and 7 days, respectively, and skin DNA was isolated 1 day after the last treatment. When DNA from exposed mice was analyzed by the 32P-postlabeling assay, 12 distinct 32P-labeled DNA adduct spots, as well as a diagonal radioactive zone, which presumably reflected the presence of incompletely resolved adducts, were noted on polyethyleneimine-cellulose TLC fingerprints. One derivative in particular (adduct 1) was seen to increase rapidly during the early treatment phase and also to persist to 8 days after treatment. The prominent adduct 1 was observed in the same location on the fingerprints of DNA samples from smokers. Cochromatography experiments suggested identity of human and mouse DNA adduct 1. Similarly, several other human and mouse adducts (adducts 3, 5, 6, and 9) appeared identical, and the diagonal radioactive zone was also present on DNA adduct maps from smokers. While absolute levels of individual human adducts were too low to be accurately quantitated, semiquantitative estimation of total tobacco-related aromatic DNA adducts in the human specimens gave values of 1 adduct in (1.7-2.9) X 10(7) nucleotides (0.10-0.18 fmol per micrograms DNA), with adduct 1 constituting 8.5-14% of the total. On the basis of these results, it appears now feasible to determine the chemical origin of smoking-induced DNA adducts in human tissues by preparation of authentic 32P-labeled reference adducts from animals treated with characterized subfractions of cigarette tar, 32P-postlabeling, and cochromatography of 32P-labeled human and animal adducts.

Correlation of aromatic hydroxylation of 11 beta-substituted estrogens with morphological transformation in vitro but not with in vivo tumor induction by these hormones.

In estrogen-induced cancer, catechol formation from administered steroids has been postulated to be a necessary event for estrogen activation and subsequent damage to cellular macromolecules. In the present study, this hypothesis has been tested using two homologous series of structurally related estrogens: estradiol, 11 beta-methylestradiol, 11 beta-ethylestradiol, 11 beta-methyl-17 alpha-ethinylestradiol, 11 beta-ethyl-17 alpha-ethinylestradiol, 11 beta-methoxy-17 alpha-ethinylestradiol, and 17 alpha-ethinylestradiol. In the Syrian hamster renal carcinoma model, only 11 beta-methylestradiol and 17 alpha-ethinylestradiol were weak carcinogens (2 of 20 and 2 of 24 hamsters with tumors, respectively). The other estrogens tested induced renal carcinoma within 6 to 9 months with an incidence in the 80-100% range. The tumor incidence in vivo did not correlate with the rates of catechol formation by hamster kidney microsomes in vitro. Compared to estradiol (relative rate, 100), catechol formation by the substituted estrogens was significantly lower, ranging from 48 (11 beta-methylestradiol) to 2 (11 beta-methoxy-17 alpha-ethinylestradiol). Kidney DNA of hamsters treated with the four 17 alpha-ethinyl estrogens, when analyzed by 32P postlabeling assay, contained the same set of covalently modified nucleotides the formation of which had previously been found to precede estrogen-induced renal carcinogenesis in vivo. In contrast, relative rates of catechol estrogen formation by BALB/c 3T3 microsomes correlated with induction of morphological transformation of BALB/c 3T3 cells and decreased in the following order: 11 beta-methylestradiol greater than 17 alpha-ethinylestradiol greater than or equal to estradiol greater than 11 beta-ethylestradiol greater than 11 beta-methoxy-17 alpha-ethinylestradiol. The hormonal potencies of several estrogen derivatives studied by various assays did not correlate with in vivo carcinogenic or in vitro cell-transforming activities. It is concluded from these experiments that in cell culture catechol formation and morphological transformation are directly related. In vivo, aromatic hydroxylation of administered estrogens did not correlate with the incidence of estrogen-induced renal carcinoma in Syrian hamsters.

Base composition studies on mitochondrial 4 S RNA from rat liver and Morris hepatomas 5123D and 7777.

The major and modified base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method. The base composition of these mitochondrial 4 S RNA preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs. The results of these studies are: 1. When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are characterized by high (A + U)/(G + C) ratios and low overall degrees of base methylation and modification. 2. The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4-S RNAs than from their cytoplasmic counterparts. Thus, several modified constituents found in both cytoplasmic and mitochondrial 4 S RNA are absent from the bacterial 4-S RNAs. 3. Mitochondrial 4S RNA from both hepatomas was found to be under-methylated and undermodified when compared with normal liver mitochondrial 4S RNA. This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepatoma 5123D (i.e., 8% undermethylation). These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors.

Base composition studies on transfer RNA from normal and regenerating rat liver.

The base composition of bulk tRNA isolated from regenerating rat liver, 12, 18, 24 and 30 h after partial hepatectomy, was determined by a 3H derivative method. Only a few minor statistically significant changes (2--11%), as compared to sham-operated liver, were found at 18, 24 and 30 h after hepatectomy. These included a reduction in the amounts of adenosine and 3-(3-amino-3-carboxypropyl)-uridine, and an increase in the amounts of 1-methyl-adenosine, 1-methylguanosine, 3-methylcytidine and pseudouridine. Similarly, when the base composition of tRNA fractions from control and 24-h regenerating rat liver, partially purified by one-dimensional polyacrylamide gel electrophoresis, was determined, no gross differences were observed. These results suggest that the process of liver regeneration is not accompanied by a gross alteration of the modification pattern of tRNA.

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.

Effects of aging and caloric restriction on I-compounds in liver, kidney and white blood cell DNA of male Brown-Norway rats.

Rodent tissues display species-, strain-, sex- and tissue-specific adduct-like DNA modifications termed I-compounds, which increase with age, are modulated by diet and are presumably derived from indigenous metabolic intermediates. We have explored whether I-compounds are affected by caloric restriction, which is known to extend life span and retard age-related degenerative and neoplastic diseases. Male Brown-Norway rats were fed NIH-31 diet ad libitum (AL). Calorically restricted (CR) rats received 60% of AL consumption, starting at 3.5 months. DNA was analyzed by 32P-postlabeling at 1, 4, 8, 12, 16 and 24 months of age in liver, kidney and white blood cells. I-compounds in AL liver and kidney exhibited complex tissue specific profiles; I-compound levels increased with age, plateaued between 8 and 18 months depending on tissue and diet and were 8.7 (liver) and 27.4 (kidney) modifications in 10(8) nucleotides at 24 months, thereby exceeding the corresponding 1-month values by 3.7- and 16.6-fold. CR resulted in similar profiles but did not diminish age-related increases, rather I-compound levels in CR liver and kidney were increased by about 70% and 30% versus age-matched AL rats. White blood cells exhibited few I-compounds and at low levels; age-related increases were small overall but more pronounced in CR rats. Higher I-compound levels in CR animals, which were presumably a consequence of metabolic effects elicited by CR, thus correlated with extended life span and, therefore, may be beneficial, in agreement with previous findings showing an association between reduced I-compound levels and hepatocarcinogenesis as well as organ susceptibility to diseases.

DNA hypomethylation in Morris hepatomas.

The 5-methylcytosine (m5C) content of DNAs from Morris hepatomas of varying growth rates and from normal liver was analyzed. DNA methylation in all hepatomas studied was found to be 20-45% less than in normal liver. This result was confirmed independently by restriction endonuclease (Hpa II and Msp I) analysis. While these results agreed with recent literature data suggesting hypomethylation of DNA from some neoplastic sources, no correlation was observed between the extent of DNA hypomethylation and the growth rates of the tumors.

12-O-tetradecanoylphorbol-13-acetate-induced rapid loss of persistent 7,12-dimethylbenz[a]anthracene-DNA adducts in mouse epidermis and dermis.

Twice-weekly application to mouse skin of 10 nmol of the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), beginning at 3 weeks after topical application of 1.2 mumol of 7,12-dimethylbenz[a]anthracene (DMBA), was found to cause a rapid loss of persistent DMBA-DNA adducts from both epidermal and dermal DNA. This effect is thought to reflect TPA-induced proliferation of quiescent initiated skin cells containing persistent adducts and may be causally related to the irreversibility of the initial phase of tumor promotion, which is known to require cell proliferation.

Highly persistent polycyclic aromatic hydrocarbon-DNA adducts in mouse skin: detection by 32P-postlabeling analysis.

A 32P-postlabeling method for carcinogen-DNA adduct analysis recently developed in our laboratory was applied to skin DNA from mice treated topically with polycyclic aromatic hydrocarbons (PAHs). After application of 4 doses of 1.2 mumol each of benzo[alpha]pyrene (BP), 3-methylcholanthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA), respectively, total covalent adduct binding in mouse skin DNA initially amounted to 1 adduct in 6.0 X 10(4) - 1.3 X 10(5) nucleotides. Four weeks after treatment, these levels had declined to 1 adduct in 1.4 X 10(6) - 2.7 X 10(6) nucleotides. Substantial removal of DNA adducts occurred during the first 2 weeks after carcinogen application while adducts remaining thereafter underwent little or no repair between 2 and 4 weeks after treatment. These results raise the possibility that the persistent adducts occupy specific genomic sites in quiescent cells where they may not be amenable to repair because of localized conformational alterations of DNA or shielding by associated proteins.

DNA damage induced by wood preserving waste extracts in vitro without metabolic activation, as assayed by 32P-postlabeling.

Aqueous wood preserving waste (WPW) extracts were tested for their ability to damage DNA in vitro without metabolic activation. Two extracts were prepared from a surface tar and a surface clay soil sample of a WPW site. As assayed by 32P-post-labelling incubation of DNA with these extracts gave rise to highly complex, extract-specific profiles of DNA adducts whose formation depended on the concentration of WPW material. Most of the adducts appeared to be derived from polycyclic aromatic hydrocarbons (PAHs). Three mg organic WPW residue gave rise to total adduct levels of 13.8 (extract 1) and 66.2 (extract 2) DNA modifications in 10(7) DNA nucleotides, corresponding to 13.9 and 26.9 modifications, respectively, per 10 mg of soil. Thus, extract 2 was more active, although the parent residue had a 1.4-times lower PAH content as determined by gas chromatography/mass spectrometry (GC/MS). DNA adduct formation presumably was a consequence of (i) free radical reactions, possibly involving semiquinones and oxygen free radicals, and (ii) reaction of direct-acting electrophiles, derived from metabolism of WPW toxicants by soil microorganisms. These reactions appeared to be more active in sample 2. The results suggest that ground water at WPW sites contains DNA-reactive compounds posing a cancer hazard to humans. The in vitro DNA adduct assay represents a novel tool to readily assess this type of hazard and the possible effects of remediation measures.

Exogenous and endogenous DNA modifications as monitored by 32P-postlabeling: relationships to cancer and aging.

32P-postlabeling analysis, a highly sensitive method for the detection and measurement of covalent carcinogen-DNA adducts and other DNA modifications, does not require radioactive test substances and, therefore, can be applied to DNA of mammals, including humans exposed to low doses of environmental or occupational genotoxicants. The basic procedure entails the enzymatic incorporation of 32P-label into hydrolysis products of DNA, followed by chromatographic mapping and autoradiography of the 32P-labeled digestion products and quantitative scintillation spectrometry. Microgram amounts of DNA are analyzed: Thus the assay is suited for limited amounts of cells or tissues. Various versions of the assay afford different sensitivities of adduct detection. A single aromatic or bulky/hydrophobic adduct in 10(8)-10(10) nucleotides can be detected and measured (corresponding to 0.3-30 amol adduct/micrograms DNA or 0.1-10 nmol adduct/mol DNA-P). In animal models, the assay has been successfully applied to a variety of mutagenic (genotoxic) as well as nonmutagenic carcinogens. In humans, DNA specimens from cigarette smokers, iron foundry workers, and coke oven workers whose total aromatic adduct levels ranged from 1 adduct in 10(6)-10(8) DNA nucleotides have been examined by 32P-postlabeling. The assay also detects DNA modifications--Indigenous (I)-compounds--that increase with age in untreated animals. I-compound profiles and levels are highly species-, strain-, sex-, and tissue-specific, and also depend on diet composition. Caloric restriction, a highly efficient method for improving resistance to carcinogenesis and extending life span, increased rather than decreased I-compound levels in various tissues of male rats. Nonmutagenic hepatocarcinogens reduced levels of I-compounds in the target organ. Because of the specificity of this effect, reduction of I-compound levels appears to represent a novel biomarker for the action of nonmutagenic carcinogens. DNA from various hepatomas was found largely devoid of I-compounds. The results support a possible antineoplastic and antiaging role of these DNA modifications.

Effects of cytochrome P450 inducers on tamoxifen genotoxicity in female mice in vivo.

We recently reported that administration of the antiestrogen tamoxifen (TAM) gives rise to two groups of DNA adducts in female mouse liver in vivo, as measured by 32P-postlabeling, and provided evidence that 4-hydroxytamoxifen and alpha-hydroxytamoxifen are proximate carcinogenic metabolites leading to group I and group II adducts, respectively (Randerath et al., Carcinogenesis 15: 2087-2094, 1994). Because cytochrome P450 (CYP) enzymes play an important role in TAM metabolism, in this investigation we tested the hypothesis that induction of liver CYP enzymes may affect TAM metabolism profoundly, resulting in increased or decreased TAM-DNA adduct formation in vivo. To this end, we treated female ICR mice with TAM either alone or in combination with one of several classic CYP inducers, i.e. phenobarbital (PB), beta-naphthoflavone (BNF), and pregnenolone-16 alpha-carbonitrile (PCN), and determined the levels of 32P-postlabeled TAM-DNA adducts and the activities of several CYP-dependent enzymes. Each of the inducers greatly diminished levels of group II, but did not affect group I adducts. TAM elicited induction of benzphetamine N-demethylase activity in liver, while activities of other enzymes were not affected. TAM, when given in combination with BNF, elicited a synergistic induction of ethoxyresorufin O-deethylase (EROD) (CYP1A1) and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities. Likewise, PCN given along with TAM caused synergistic induction of EROD and ethylmorphine N-demethylase activities. There was no synergism between PB and TAM, however. Overall, the results further support the existence of two pathways of TAM metabolism to DNA-reactive electrophiles and strongly suggest that the classic CYP inducers tested enhance detoxication of TAM to non-genotoxic metabolites.

Postlabeling methods for carcinogen-DNA adduct analysis.

Radioactive carcinogens have provided most of our present knowledge about the chemistry of interactions between carcinogens and biological systems. The requirement of radioactive carcinogens has restricted carcinogen-DNA binding studies to chemicals that are readily available in isotopically labeled form, i.e., a minute fraction of all potentially mutagenic or carcinogenic chemicals. To extend the scope of carcinogen-DNA binding studies, an alternative method, which does not require radioactive test chemicals, has been developed. In this approach, radioactivity (32P) is being incorporated into DNA constituents by polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP after exposure of the DNA in vitro or in vivo to a nonradioactive, covalently binding chemical, and evidence for the alteration of DNA nucleotides is provided by the appearance of extra spots on autoradiograms of thin-layer chromatograms of digests of the chemically modified DNA. Quantitation of adduct levels is accomplished by scintillation counting. The sensitivity of the technique depends on the experimental conditions for 32P-labeling and on the chemical structure of the adducts. Greater sensitivity may be achieved if adducts can be separated as a class from the normal nucleotides. This is the case for an estimated 80% of all carcinogens, giving rise to bulky and/or aromatic substituents in DNA. Under the present conditions, one such adduct in 10(9) to 10(10) normal nucleotides can be detected. A total of approximately 80 compounds has been studied thus far Binding to DNA of rodent tissues was readily detected by the 32P-postlabeling assay for all known carcinogens among these compounds, and adducts were detected in DNA from human placenta of smokers.