Guo-Dong Zhou

Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts.

The detection of 1,N2-propanodeoxyguanosine adducts in the DNA of rodent and human tissues as endogenous lesions has raised important questions regarding the source of their formation and their roles in carcinogenesis. Both in vitro and in vivo studies have generated substantial evidence which supports the involvement of short- and long-chain enals derived from oxidized polyunsaturated fatty acids (PUFAs) in their formation. These studies show that: (1) the cyclic propano adducts are common products from reactions of enals with DNA bases; (2) they are formed specifically from linoleic acid (LA; omega-6) and docosahexaenoic acid (omega-3) under in vitro stimulated lipid peroxidation conditions; (3) the levels of propano adducts are dramatically increased in rat liver DNA upon depletion of glutathione; (4) the adduct levels are increased in the liver DNA of the CCl4-treated rats and the mutant strain of Long Evans rats which are genetically predisposed to increased lipid peroxidation; and (5) adduct levels are significantly higher in older rats than in newborn rats. These studies collectively demonstrate that tissue lipid peroxidation is a main endogenous pathway leading to propano adduction in DNA. The possible contribution from environmental sources, however, cannot be completely excluded. The mutagenicity of enals and the mutations observed in site-specific mutagenesis studies using a model 1,N2-propanodeoxyguanosine adduct suggest that these adducts are potential promutagenic lesions. The increased levels of the propano adducts in the tissue of carcinogen-treated animals also provide suggestive evidence for their roles in carcinogenesis. The involvement of these adducts in tumor promotion is speculated on the basis that oxidative condition in tissues is believed to be associated with this process.

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene.

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.

A 32P-Postlabeling Assay for the Oxidative DNA Lesion 8,5′-Cyclo-2′-deoxyadenosine in Mammalian Tissues EVIDENCE THAT FOUR TYPE II I-COMPOUNDS ARE DINUCLEOTIDES CONTAINING THE LESION IN THE 3′ NUCLEOTIDE

8,5′-Cyclopurine-2′-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of 32P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5′-cyclo-2′-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic32P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The 32P-postlabeling technique used here is capable of detecting 1–5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.

Bulky endogenous DNA modifications (I-compounds)—possible structural origins and functional implications

I-compounds are bulky covalent DNA modifications which increase with age in tissues of unexposed laboratory animals and are derived from endogenous DNA-reactive intermediates of nutrient and oxygen metabolism. They have been classified into 2 major groups, i.e., type I and type II. Profiles and levels of type I I-compounds show considerable variation depending on species, strain, tissue, and gender, but are also affected by diet and chemical and hormonal exposures, indicating their formation to be determined by genetic and environmental factors. For example, sex hormones, dietary oat lipids, and isoprenoids affect their profiles and/or levels in tissue DNA. A gradual depletion of many type I I-compounds occurs during carcinogenesis, as many carcinogens/tumor promoters significantly reduce their levels, and neoplasms display very low levels, apparently independent of growth rate, indicating a loss of the ability to form these modified nucleotides. Conversely, dietary restriction, the most effective method to retard carcinogenesis and aging, significantly elevates type I I-compound levels, as compared to age-matched ad libitum-fed animals. Levels of many liver and kidney I-compounds exhibit genotype- and diet-dependent positive linear correlations with median life span. Formation of high levels of oat-related type I I-compounds has been associated with reduced formation of carcinogen-induced preneoplastic hepatic foci. These results suggest that such DNA modifications may not represent DNA lesions but may rather be functionally important. This view is supported by circadian rhythms displayed by some I-compounds. Thus, certain type I I-compounds may play a protective role against carcinogenesis and age-associated degenerative processes. Type II I-compounds, on the other hand, represent DNA damage and include several bulky lesions, which are enhanced by pro-oxidant carcinogens such as ferric nitrilotri- acetate (Fe-NTA) in target organ (kidney) DNA of rodents and are identical to products generated by oxidizing DNA or oligonucleotides under Fenton reaction conditions in vitro. Some of these products appear to be base-base or base-sugar intrastrand crosslinks. Notably, Fe-NTA reduces the levels of type I I-compounds in renal DNA. Type II I-compound levels are increased in tissue DNA of normal newborn rats. The formation of oxidative DNA lesions in neonates is most likely caused by oxidative stress associated with the sudden increase of partial oxygen pressure in arterial blood and tissues at birth. In view of the rapid cell replication at this developmental stage, endogenous oxidative DNA lesions sustained early in life may contribute to the development of cancer and degenerative diseases later in life.

Genotoxicity of complex PAH mixtures recovered from contaminated lake sediments as assessed by three different methods

Although human exposure generally occurs to mixtures of chemicals, limited toxicological information is available to characterize the potential interactions of the components of environmental mixtures. This study was conducted to compare the genotoxicity of chemically characterized polycyclic aromatic hydrocarbon (PAH) mixtures using in vitro and in vivo techniques. A total of three extracts (E1–E3) were selected from sediment samples collected from a lake adjacent to an abandoned coal gasification site. Sediments were collected on a grid moving downstream and away from the most likely source of PAH contamination, with E1 collected closest to the shore, E2 at an intermediate distance, and E3 furthest from the shore. The sediment samples were extracted in methylene chloride and methanol, dried, and redissolved in an appropriate solvent for evaluation in a battery of genotoxicity assays. Samples were evaluated for their ability to produce point mutations in bacteria and DNA adducts in vitro without metabolic activation or in vivo. Samples were also analyzed using GC/MS. Sample E1 had both the highest concentration of benzo(a)pyrene (BP) (46.5 ppm) and carcinogenic PAHs and, using 32P‐postlabeling, induced the highest adduct levels overall in vitro and in vivo. Sample E2, which had a BP concentration of 14 ppm, induced the greatest number of revertants in the bacterial mutagenicity assay. Sample E3, which had the lowest level of carcinogenic PAHs and BP, induced the lowest adduct levels. However, E3 was capable of inducing a positive genotoxic response in bacteria (with S9), although the slope of the response at lower doses was less than that of E2. The in vivo data showed that the major adduct formed by E1 and E2 was a BP adduct. This information could not have been obtained with the Salmonella or in vitro postlabeling tests. Among internal organs, the extracts of all three samples induced the greatest adduct levels in the lung, similarly to previous complex PAH mixtures studied. These data demonstrate the limitations of predicting genotoxic or carcinogenic potential based on chemical analysis or a single biological test. The results suggest that mixture interactions, cytotoxicity and metabolism are likely to have an influence on the potential of a complex mixture of chemicals to produce a carcinogenic effect. In addition, the concentration of genotoxic PAHs and both in vitro and in vivo DNA adduct formations were decreased with increasing distance from the shoreline. Environ. Mol. Mutagen. 33:303–312, 1999

Biomarkers of aging: correlation of DNA I-compound levels with median lifespan of calorically restricted and ad libitum fed rats and mice

I-compounds are species-, tissue-, genotype-, gender-, and diet-dependent bulky DNA modifications whose levels increase with animal age. While a few of these DNA modifications represent oxidation products, the majority of I-compounds appear to be derived from as yet unidentified endogenous DNA-reactive intermediates other than reactive oxygen species. Circadian rhythms of certain I-compounds in rodent liver imply that levels of these DNA modifications are precisely regulated. Caloric restriction (CR), the currently most effective method available to retard aging and carcinogenesis, has been previously shown to elicit significant elevations of I-compound levels in tissue DNA from Brown-Norway (BN) and F-344 rats as compared to age-matched ad libitum fed (AL) animals. The present investigation has extended this work by examining liver and kidney DNA I-compound levels in three genotypes of rats (F-344, BN, and F-344 x BN) and two genotypes of mice (C57BL/6N and B6D2F1) under identical experimental conditions in order to determine whether correlations exist between I-compound levels, measured in middle-aged animals, and median lifespan. Levels of a number of liver and kidney I-compounds were found to display genotype- and diet-dependent, statistically significant positive linear correlations with median lifespan in both species. In particular, the longer-lived hybrid F-344 x BN rats and B6D2F1 mice tended to exhibit higher I-compound levels than the parent strains. CR enhanced I-compound levels substantially in both rats and mice. Thus, I-compounds, measured at middle age, reflected the functional capability (health) of the organism at old age, suggesting their predictive value as biomarkers of aging. The positive linear correlations between levels of certain I-compounds (designated as type I) and lifespan suggest that these modifications may be functionally important and thus not represent endogenous DNA lesions (type II), whose levels would be expected to correlate inversely with lifespan.

Enhancement of age-related increases in DNA I-compound levels by calorie restriction: comparison of male B-N and F-344 rats

Caloric restriction (CR), known to extend median and maximum life spans, improve resistance to carcinogenesis, and significantly retard age-associated degenerative diseases in rodents, was previously reported to modulate levels of indigenous, age-dependent DNA modifications, called I-compounds, in male Brown-Norway (B-N) rats. Since profiles of these adduct-like derivatives are species-, strain-, sex-, and tissue-specific, we explored this apparent CR/I-compound relationship in a comparative study between male B-N and male Fischer 344 (F-344) rats, the latter having a shorter life expectancy and high incidence of renal disease. Control animals were fed NIH-31 diet ad libitum (AL), while the caloric intake of CR animals was limited to 60% of AL, starting at 3.5 months. Liver and kidney DNA from 1, 8, 12, 16, 24 (AL, CR), and 30 (CR only) month old rats was analyzed by 32P-postlabeling. Corresponding tissues from the two strains yielded similar DNA profiles. Total liver I-compound levels displayed 2.3-4.6-fold age-dependent increases from 1 to 24 months, and kidney values at 24 months were 5.2-8 times higher than those at 1 month. In both strains, I-compound levels of CR animals were higher, up to 2-fold, than in age-matched AL rats. Regression analyses indicated linear relationships between most CR relative adduct labeling values (both total and individual fractions) and age, whereas many AL values exhibited this type of link with log age. These findings confirm that a correlation exists between CR and I-compound levels, and, given the above physiological benefits of CR, indicate that I-compounds represent biomarkers of aging with potential utility in intervention studies.

Intensification and depletion of specific bulky renal DNA adducts (I-compounds) following exposure of male F344 rats to the renal carcinogen ferric nitrilotriacetate (Fe-NTA).

The effects of the renal carcinogen ferric nitrilotriacetate (Fe-NTA) on kidney DNA of male F344 rats were studied to determine whether bulky DNA oxidation products (putative intrastrand crosslinks) could be detected by 32P-postlabeling in the target organ of carcinogenesis. Rats (10-11 weeks old) were given a single dose of Fe-NTA (15 mg Fe/kg body weight) i.p. at 3:00 pm. After 5 h, renal DNA from Fe-NTA-treated and vehicle control animals was assayed by 32P-postlabeling. Thin-layer chromatography and quantitative analysis of two labeled nucleotide fractions of increasing polarity, L and C, showed that three spots (L1, L2, and C3) were intensified 3.5- to 4.2-fold in treated animals. L1 consisted of subfractions L1a, L1b, and L1c, which could be resolved chromatographically. L1c, L2, and C3 were identical to DNA oxidation products generated by the Fenton reaction in vitro, while L1a and L1b apparently did not arise by this mechanism. DNA damage and toxicity appeared reduced in younger animals and animals treated in the morning, presumably due to differences in antioxidant defenses. Liver and lung (non-target organs) DNA did not exhibit enhanced L1, L2, and C3 spots. In addition to augmenting renal I-compounds, Fe-NTA reduced the levels of three major polar kidney I-compounds (C4, C5, and C6) to 22-53% of control. This reduction did not appear to arise by direct oxidative DNA damage, resembling the previously documented loss of liver I-compounds induced by numerous hepatocarcinogens. Two of these I-compounds (C4 and C5) have been reported to exhibit positive linear correlations with median lifespan of male F344 rats. The pleiotropic response of kidney I-compound levels to Fe-NTA was consistent with different roles of different types (I and II) of I-compounds in Fe-NTA-mediated renal carcinogenesis. The results strongly support a causal relationship between oxidative DNA lesions and Fe-NTA-mediated carcinogenesis.

Acute Elevation by Short-Term Dietary Restriction or Food Deprivation of Type I I-Compound Levels in Rat Liver DNA

Type I I-compounds are bulky endogenous DNA modifications detectable by 32P postlabeling that exhibit age, species, tissue, genotype, gender, and diet dependence. Their formation appears unrelated to oxidative stress. In fact, several lines of indirect evidence suggest that many type I I-compounds may represent normal functional DNA modifications. For example, long-term dietary restriction (DR), which retards the development of age-related diseases including cancer and extends median and maximum life spans, unexpectedly elicits significant increases rather than decreases in the levels of many I-compounds in different rodent tissues. Positive linear correlations have been observed between such levels and median life spans of the animals. In the present work we have investigated 1) whether elevation of I-compound levels does not depend on chronic DR, i.e., occurs after a short period of DR or fasting, and 2) whether I-compound levels return to control values after the animals are returned to unrestricted feeding after food deprivation. Female Fischer 344 rats (approx 140 g each) were randomized into three groups. Group I was fed a natural ingredient (Purina 5001) diet ad libitum (AL) throughout the study, Group 2 was switched to 60% of the AL amount (40% DR) at 0 hour, and Group 3 was given no food for up to 72 hours and then returned to AL feeding until the end of the experiment. Liver DNA of individual rats (n = 4) was isolated for I-compound analysis at 24, 72, and 240 hours. Restricted and food-deprived rats showed elevated levels of hepatic I-compounds, with fasting eliciting the highest levels. These effects were seen as early as the 24-hour time point. Refeeding after 72 hours of food deprivation restored the levels to control values, measured at 240 hours. Our observations are discussed in relation to carcinogenesis and tumor promotion. The almost instantaneous changes of endogenous DNA modifications showed their exquisite sensitivity to nutritional factors and provided strong new evidence for precise regulation of their formation and removal.

Effects of dietary transition metals on oxidative DNA lesions in neonatal rats

Bulky endogenous oxidative lesions (type II I-compounds) reflect DNA damage associated with oxidative stress. As shown by 32P-postlabeling, their levels are enhanced by pro-oxidant genotoxins and also shortly after normal birth in several rat tissues as a function of time and the maternal diet. In order to elucidate which dietary components contribute to postnatal DNA damage, we have focused, herein, on the possible role of transition metals (iron, copper, and nickel). Pregnant Fischer 344 (F344) rats were fed AIN-93G purified diet containing different amounts of iron, copper, and nickel, or Purina-5001 natural-ingredient diet (which contains relatively high concentrations of these metals). Type II I-compounds were estimated by nuclease P1-enhanced 32P-postlabeling in liver and lung DNA of fetuses and at 24h and day 9 post-partum. Increased postnatal oxidative damage was detected in liver but not lung DNA of neonates exposed to higher amounts of dietary transition metals. There were significant positive linear correlations between maternal transition metal intake and neonatal, but not fetal and maternal type II I-compound levels. The results show that transition metals in the maternal diet affect perinatal oxidative DNA damage, presumably via a Fenton-type reaction. They also provide evidence for optimal levels in the maternal diet of transition metals, which on one hand, are essential for life, but on the other, can cause potentially deleterious DNA alterations in the offspring.