Oxidative damage and direct adducts in calf thymus DNA induced by the pentachlorophenol metabolites, tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone.

DNA damage induced by quinoid metabolites of pentachlorophenol (PCP), i.e. tetrachloro-1,4-benzoquinone (Cl(4)BQ) and tetrachlorohydroquinone (Cl(4)HQ), was investigated in calf thymus DNA. The (32)P-post-labeling assay revealed four major and several minor adducts (3.5 adducts per 10(5) total nucleotides) that were produced in calf thymus DNA treated with Cl(4)BQ (5 mM). These DNA adducts were chemically stable even after conditions that induce thermal depurination and are unlikely to undergo depurination/depyrimidination to form apurinic/apyrimidinic (AP) sites. In addition, increases in 8-hydroxy-deoxyguanosine (8-HO-dG) (5 8-HO-dG per 10(5) nucleotides) and AP sites (0.5 AP sites per 10(5) nucleotides) were observed in Cl(4)BQ-modified calf thymus DNA. Further investigation indicated that in the presence of Cu(II) and NADPH, low concentrations of Cl(4)BQ (1 microM) induced a doubling of 8-HO-dG (10 8-HO-dG per 10(5) nucleotides) and dramatic increases in AP sites (20 AP sites per 10(5) nucleotides) and DNA single-strand breaks. The types of DNA damage induced by Cl(4)HQ plus Cu(II) were similar to those by Cl(4)BQ plus Cu(II) and NADPH, whereas catalase inhibited the formation of DNA damage. These data suggest that oxidative damage is causally involved in the formation of AP sites. Concentration-dependent increases in 8-HO-dG induced by Cl(4)HQ plus Cu(II) and Cl(4)BQ plus Cu(II) and NADPH were correlated with the formation of AP sites (r(2) = 0.977) with a ratio of 8-HO-dG to AP sites at 1:1.6. The AP site-cleavage assay confirmed that approximately 85% of the AP sites induced by Cl(4)HQ and Cu(II) were detected as 5'-cleaved AP sites. Since hydrogen peroxide alone causes similar DNA damage, these results suggest the involvement of Cu(II) and hydrogen peroxide in the induction of oxidative DNA damage by Cl(4)HQ/Cl(4)BQ. The data demonstrate that PCP quinone and hydroquinone induce direct and oxidative base modifications as well as the formation of 5'-cleaved AP sites in genomic DNA. These lesions may have important implications for PCP clastogenicity and carcinogenicity.

Induction of direct adducts, apurinic/apyrimidinic sites and oxidized bases in nuclear DNA of human HeLa S3 tumor cells by tetrachlorohydroquinone.

DNA damage induced by tetrachlorohydroquinone (Cl(4)HQ), the quinonoid metabolite of pentachlorophenol (PCP), was investigated in human HeLa S3 tumor cells. Formation of one major and two minor DNA adducts in cells treated with Cl(4)HQ (50-300 microM) was detected by (32)P-post-labeling assay and the adducts accumulated over the course of the experiment (0.5-2 h), with total adduct levels estimated to be 3-6 per 10(8) nucleotides. These adducts did not correspond to those derived from calf thymus DNA treated with tetrachloro-1,4-benzoquinone. Results from the apurinic/apyrimidinic (AP) sites assay indicated that the number of AP sites was 2-fold greater in cells exposed to Cl(4)HQ (300 microM) than the corresponding control. Further characterization of the AP sites confirmed that Cl(4)HQ induced predominantly (75%) putrescine-excisable AP sites in HeLa S3 cells. In parallel, the concentration of 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in cells treated with Cl(4)HQ for 0.5 and 2 h was increased 2- and 5-fold, respectively, compared with the control. The extent of oxidative DNA damage induced by Cl(4)HQ was approximately two orders of magnitude greater than those of direct DNA adducts. Overall, it appears that reactive oxygen species mediate the parallel formation of AP sites and 8-HO-dG in HeLa S3 cells following treatment with Cl(4)HQ and that the contribution of depurination/depyrimidination of direct DNA adducts is relatively insignificant compared with the formation of oxidized AP sites. We conclude that putrescine-excisable AP sites represent a major type of ROS-mediated oxidative DNA damage in cellular DNA induced by Cl(4)HQ and may play a role in PCP-induced clastogenicity in mammalian cells.

Formation of quinonoid-derived protein adducts in the liver and brain of Sprague-Dawley rats treated with 2,2',5, 5'-tetrachlorobiphenyl.

A possible role for metabolic activation of 2,2',5, 5'-tetrachlorobiphenyl (TCB) to quinonoid metabolites was investigated in vitro in rat liver microsomes and in vivo in male Sprague-Dawley rats. Incubation of TCB with phenobarbital-induced rat liver microsomes resulted in metabolism of TCB to 3-hydroxy-TCB (3-OH-TCB) and 3,4-dihydroxy-TCB (3,4-diOH-TCB), which were further oxidized to form a reactive intermediate that bound to liver proteins. The predominant species observed in the Raney nickel assay for cysteinyl adducts was identified as 3,4-diOH-TCB, consistent with an adduct having the structure 5-cysteinyl-3,6-dichloro-4-(2', 5'-dichlorophenyl)-1,2-benzoquinone. This adduct may arise via the Michael addition of the sulfhydryl group of cysteine to 3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone (Cl(4)PhBQ). Metabolism of 3-OH-TCB by phenobarbital-induced microsomes in the presence of either NADPH or cumene hydroperoxide as a cofactor resulted in the formation of adducts. Dose-dependent formation of cysteinyl adducts was observed in liver cytosolic protein from rats treated with a single dose of TCB (0-200 mg/kg) by gavage. By regression analysis, the TCB adducts decayed with a half-life of 2. 03 +/- 0.131 days (mean +/- SE), which is approximately 2.5-fold shorter than the endogenous half-life for liver cytosolic protein in rat liver, suggesting adduct instability. Saturable formation of TCB adducts was observed in liver cytosolic protein of rats receiving multiple doses of TCB over 5 days. The levels of Cl(4)PhBQ-derived adducts were 2.1-fold greater than the estimated steady-state levels predicted by the single-dose treatment [97.7 +/- 13.2 vs 45.7 +/- 3. 73 (pmol/g)/(mg/kg of body weight)], suggesting induction of metabolism. A single cysteinyl adduct, inferred to be 5-cysteinyl-3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone, was detected in brain cytosolic protein of rats treated with multiple doses of TCB with levels of 15.2 (pmol/g)/(mg/kg of body weight). Implied involvement of a reactive quinone in the liver and brain of TCB-treated rats supports the idea that quinonoid metabolites may be important contributors to PCB-derived oxidative damage to genomic DNA.

5'-nicked apurinic/apyrimidinic sites are resistant to beta-elimination by beta-polymerase and are persistent in human cultured cells after oxidative stress.

Genomic DNA is continuously exposed to oxidative stress. Whereas reactive oxygen species (ROS) preferentially react with bases in DNA, free radicals also abstract hydrogen atoms from deoxyribose, resulting in the formation of apurinic/apyrimidinic (AP) sites and strand breaks. We recently reported high steady-state levels of AP sites in rat tissues and human liver DNA (Nakamura, J., and Swenberg, J. A. (1999) Cancer Res. 59, 2522-2526). These AP sites were predominantly cleaved 5' to the lesion. We hypothesized that these endogenous AP sites were derived from oxidative stress. In this investigation, AP sites induced by ROS were quantitated and characterized. A combination of H(2)O(2) and FeSO(4) induced significant numbers of AP sites in calf thymus DNA, which were predominantly cleaved 5' to the AP sites (75% of total aldehydic AP sites). An increase in the number of 5'-AP sites was also detected in human cultured cells exposed to H(2)O(2), and these 5'-AP sites were persistent during the post-exposure period. beta-Elimination by DNA beta-polymerase efficiently excised 5'-regular AP sites, but not 5'-AP sites, in DNA from cells exposed to H(2)O(2). These results suggest that 5'-oxidized AP sites induced by ROS are not efficiently repaired by the mammalian short patch base excision repair pathway.

The effects of exposure route on DNA adduct formation and cellular proliferation by 1,2,3-trichloropropane.

1,2,3-Trichloropropane (TCP) induces high incidences of tumors at multiple sites in mice and rats when administered chronically by gavage. The animal tumor data are being used to predict human risk from potential exposure to TCP in drinking water. Risk assessment may be affected by differences in the route of exposure. Gavage administration, which results in high bolus concentrations compared to drinking water exposure, may quantitatively affect toxicokinetics, cytotoxicity, and genotoxicity. We have examined the effects of TCP exposure by the two routes on the formation of DNA adducts and the induction of cellular proliferation. Male B6C3F1 mice were administered [14C]TCP for 1 week by gavage or in drinking water at the low dose (6 mg/kg) used in the NTP carcinogenesis bioassay. Two target organs (forestomach and liver) and two nontarget organs (glandular stomach and kidney) were examined for DNA adduct formation. Adducts were hydrolyzed from DNA, isolated by HPLC, and quantitated by measuring HPLC fractions for radioactivity. In the forestomach, liver, and kidney, gavage administration of TCP resulted in 1.4-to 2.4-fold greater yields of the major DNA adduct, previously identified as S-[1-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione. Significant differences in cell proliferation, as determined by incorporation of bromodeoxyuridine into DNA, were also observed for the two routes. Gavage administration of TCP for 2 weeks resulted in up to a threefold greater cell proliferation rate relative to administration in drinking water. Our findings of exposure-related differences in TCP-induced DNA adduct formation and cell proliferation suggest that a risk assessment based on the existing gavage study may overestimate human risk.

DNA adducts: biological markers of exposure and potential applications to risk assessment.

DNA adducts have been investigated extensively during the past decade. This research has been advanced, in part, by the development of ultrasensitive analytical methods, such as 32P-postlabeling and mass spectrometry, that enable detection of DNA adducts at concentrations as low as one adduct per 10(9) to 10(10) normal nucleotides. Studies of mutations in activated oncogenes such as ras, inactivated tumor suppressor genes such as p53, and surrogate genes such as hprt provide linkage between DNA adducts and carcinogenesis. The measurement of DNA adducts, or molecular dosimetry, has important applications for cancer risk assessment. Cancer risk assessment currently involves estimating the probable effects of carcinogens in humans based on results of animal bioassays. Estimates of risk are then derived from mathematical models that fit data of tumor incidence at the high animal exposures and extrapolate to probable human exposures that may be orders of magnitude lower. Molecular dosimetry could extend the observable range of mechanistic data several orders of magnitude lower than can be achieved in carcinogenesis bioassays. This measurement also compensates automatically for individual and species differences in toxicokinetic factors, as well as any nonlinearities that affect the quantitative relationships between exposure and molecular dose. As a result, molecular dosimetry can provide a basis for conducting high- to low-dose, route-to-route, and interspecies extrapolations. The incorporation of such data into risk assessment promises to reduce uncertainties and produce more accurate estimates of risk compared to current methods.

Hemoglobin and DNA adduct formation in Fischer-344 rats exposed to 2,4- and 2,6-toluene diamine.

Using gas chromatography/mass spectrometry for detection of hemoglobin adducts, and 32P-postlabelling for DNA adducts, we examined macromolecular binding in Fischer-344 rats administered 2,4-or 2,6-toluene diamine (TDA). The dose-response and correlative relationship between the two macromolecules were investigated over a range of doses (0-250 mg/kg). The time course of adduct formation and removal was also examined. Both TDA isomers induced formation of hemoglobin adducts, but only the 2,4-isomer induced DNA binding. Maximum hemoglobin and DNA adduct levels were detected 24 h following administration. Both hemoglobin and DNA binding increased in a dose-dependent manner. Hemoglobin adduct clearance demonstrated a nonlinear decay, with adduct loss occurring faster than normal erythrocyte clearance. The effects of metabolic inhibitors on adduct formation were examined using piperonyl butoxide and pentachlorophenol to inhibit p450 isozymes and sulfotransferase, respectively. Microsomal enzymatic activation was critical to hemoglobin adduct formation with inhibition by piperonyl butoxide reducing adduct yields by over 90%. Sulfation did not appear to play a significant role in TDA-induced hemoglobin adduct formation.

Dose-response relationships for carcinogens.

Biotransformation of chemical carcinogens involves both metabolic activation and detoxication. The molecular dose present on DNA as adducts represents a balance between these two pathways (formation) and DNA repair. All of these are enzymatic processes subject to saturation. When none of the pathways is saturated, linear molecular dosimetry is expected, whereas if metabolic activation is saturated, a supralinear response occurs. If detoxication or DNA repair is saturated, a sublinear response occurs. With chronic exposure, steady-state concentrations of DNA adducts develop and these follow the same patterns. With several alkylating agents, multiple adducts are formed. The extent of formation is chemically defined, but different DNA repair pathways can be involved for different adducts. By understanding the molecular dose and biology of each adduct and comparing these to the dose-response for tumor induction, it may be possible to identify the most appropriate biomarkers for risk assessment. Recently, endogenous DNA adducts identical to those induced by known human carcinogens have been identified. These endogenously formed adducts may play an important role in human carcinogenesis.

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1,2,3-trichloropropane.

1,2,3-Trichloropropane (TCP) is a multispecies, multisite carcinogen which has been found to be an environmental contaminant. In this study, we have characterized and measured DNA adducts formed in vivo following exposure to TCP. [14C]TCP was administered to male B6C3F1 mice and Fischer-344 rats by gavage at doses used in the NTP carcinogenesis bioassay. Both target and nontarget organs were examined for the formation of DNA adducts. Adducts were hydrolyzed from DNA by neutral thermal or mild acid hydrolysis, isolated by HPLC, and detected and quantitated by measurement of radioactivity. The HPLC elution profile of radioactivity suggested that one major DNA adduct was formed. To characterize this adduct, larger yields were induced in rats by intraperitoneal administration of TCP (300 mg/kg). The DNA adduct was isolated by HPLC based on coelution with the radiolabeled adduct, and compared to previously identified adducts. The isolated adduct coeluted with S-[1-(hydroxymethyl)-2-(N7-guanyl)-ethyl]glutathione, an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray mass spectrometry suggested that the TCP-induced adduct and the DBCP-derived adduct were identical. The 14C-labeled DNA adduct was distributed widely among the organs examined. Adduct levels varied depending on species, organ, and dose. In rat organs, adduct concentrations for the low dose ranged from 0.8 to 6.6 mumol per mol guanine and from 7.1 to 47.6 mumol per mol guanine for the high dose. In the mouse, adduct yields ranged from 0.32 to 28.1 mumol per mol guanine for the low dose and from 12.2 to 208.1 mumol per mol guanine for the high dose. The relationship between DNA adduct formation and organ-specific tumorigenesis was unclear. Although relatively high concentrations of DNA adducts were detected in target organs, several nontarget sites also contained high adduct levels. Our data suggest that factors in addition to adduct formation may be important in TCP-induced carcinogenesis.

Formation and removal of DNA adducts in Fischer-344 rats exposed to 2,4-diaminotoluene.

32P-Postlabeling was used to examine DNA adduct formation and removal in Fischer-344 rats exposed to the animal carcinogen 2,4-diaminotoluene (DAT). Adduct formation and persistence were compared between target (liver and mammary gland) and non-target organs (kidney and lung) to determine if possible differences could explain the observed organ specificity of DAT induced carcinogenesis. The effects of different exposure conditions on DNA adduct formation and removal were also examined by varying the concentration and frequency of compound administration. DAT produced three distinct DNA adducts. Among the organs examined, DNA binding was highest in the liver, with levels approximately 10 times greater than that of the mammary gland and up to 50 times greater than of the two nontarget sites. Despite the large differences in the initial extent of adduct formation, the persistence of adducts among sites was not significantly different. In the liver, there were dose-dependent differences in DNA adduct formation, but adduct removal following different dosages did not vary significantly. The effects of multiple administration on DNA adduct formation and removal were examined by treating rats with 5 mg/kg DAT daily for 10 consecutive days. Adduct yields from multiple treatment were greater than from a single 50 mg/kg exposure. The persistence of adducts following multiple treatment was also greater than after an equivalent single exposure. The results demonstrated organ-specific and dose-dependent differences in initial extent of DNA adduct formation, but no differences in adduct persistence. However, the results did suggest that adduct formation and persistence may change with repeated administration of DAT.

Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene.

We used 32P-postlabelling to compare DNA binding between the potent hepatocarcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene. The two compounds were compared to determine whether differences in DNA binding could partly explain the differences in their carcinogenicity. Fischer-344 rats were administered 1.2 mmol/kg of a compound by single i.p. injection and examined for DNA adduct formation in the liver. Four adducts were detected following administration of 2,6-dinitrotoluene, with a total adduct yield of 13.5 adducted nucleotides per 10(7) nucleotides. Qualitatively identical adducts were also detected after treatment with the derivative 2-amino-6-nitrotoluene. Adduct yields from 2,6-dinitrotoluene were 30 times greater than from 2-amino-6-nitrotoluene. No adducts were observed following treatment with 2,6-diaminotoluene. 2,6-Dinitrotoluene and 2,6-diaminotoluene were also compared for qualitative differences in hepatotoxicity. 2,6-Dinitrotoluene produced extensive hemorrhagic necrosis in the liver, whereas no evidence of hepatocellular necrosis was detected following administration of the latter. The differences between the two compounds in both DNA binding and cytotoxicity were consistent with the differences in their carcinogenicity.

32P-postlabelling analysis of DNA adducts from Fischer-344 rats administered 2,4-diaminotoluene.

Using 32P-postlabelling and thin layer chromatography, DNA adduct formation by the potent animal carcinogen 2,4-diaminotoluene in Fischer-344 rats was investigated. DNA from four different organs, liver, mammary gland, kidney and lung, were examined for adducts following single administration of this compound. DNA binding was detected in all four organs, with each producing one major and two minor adduct spots on autoradiograms. The adducts induced were qualitatively identical among the different organs, but quantitative differences were observed. The two target organs of 2,4-diaminotoluene induced carcinogenesis, the liver and mammary gland produced higher adduct yields, with levels up to 30-times higher than those for the two non-target organs. Since the liver is the principal target for 2,4-diaminotoluene induced carcinogenesis, we further examined DNA adducts from this site for the effects of different doses and time points. DNA binding in liver was detected following doses as low as 4.1 mumol/kg. At the highest concentration examined (2046 mumol/kg), the level of the major adduct was 29.2 adducted nucleotides per 10(7) total nucleotides. The yields for the two minor adducts were approximately one-tenth that for the major adduct. Following a 410 mumol/kg dose, DNA adduct removal over time was examined. DNA adduct removal exhibited biphasic kinetics, with a rapid initial phase followed by a slower rate of elimination. Up to 60% of maximum adduct levels persisted after 2 weeks. DNA binding by 2,4-diaminotoluene was also compared to that by its weakly carcinogenic analog, 2,4-dinitrotoluene. The two compounds produced identical adduct patterns, suggesting that they share common metabolites and adducts. Adduct yields from 2,4-dinitrotoluene, however, were lower. The results of our studies suggest that the differences in carcinogenic potency between 2,4-diaminotoluene and 2,4-dinitrotoluene, as well as the organotropic effects of 2,4-diaminotoluene may be explained, in part, by quantitative differences in the extent of DNA adduct formation.

Comparison of DNA adduct formation between 2,4 and 2,6-dinitrotoluene by 32P-postlabelling analysis.

Using 32P-postlabelling, we examined DNA binding by 2,4 and 2,6-dinitrotoluene (DNT) in Fischer-344 rats. DNA binding between the two compounds was compared to determine if differences in adduct formation and persistence could partly explain the known isomer-specific hepatocarcinogenicity of DNTs. The differences in cytotoxicity between the two isomers were also assessed. Both 2,4 and 2,6-DNT induced adduct formation in hepatic DNA. Three distinct adducts were detected following single i.p. administration of 2,4-DNT, while the 2,6-isomer produced four different adducts. Depending on the concentration administered, the two compounds differed in their relative yields. 2,6-DNT produced a greater total adduct yield relative to the 2,4-isomer at low concentrations. Following administration of high concentrations, however, 2,4-DNT predominated. The maximum adduct levels measured were 3.0 and 1.8 adducted nucleotides per 10(6) nucleotides for 2,4 and 2,6-DNT, respectively. Substantial amounts of adducts from both compounds were found to persist over time. After 2 weeks, the mean persistence for 2,4 and 2,6-DNT induced adducts were 42% and 46%, respectively. Qualitative examination for liver toxicity showed 2,6-DNT to be more cytotoxic, inducing extensive hemorrhagic centrilobular necrosis. Rats treated with 2,4-DNT did not show any observable signs of hepatocellular necrosis. Under the conditions of this study, the differences between 2,4 and 2,6-DNT in adduct formation and persistence do not appear to be sufficient to account for their differences in carcinogenicity. The toxicity of 2,6-DNT may be a determining factor in the potent carcinogenicity observed with this compound.