The deacetylation of N-hydroxy-2-acetylaminofluorene by rat liver microsomes and carboxyl esterase

We have demonstrated that incubation of rat liver microsomes with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) leads to formation of a 2-nitrosofluorene-membrane lipid adduct. This adduct exists as a nitroxyl free radical, termed N-O-LAF, in its oxidized state. When microsomes were incubated with the sulfhydryl binding agent, rho-hydroxymercuribenzoate, a larger amount of N-OL-LAF formed. We interpret this as a slowdown in the rate of endogenous chemical reduction of carcinogen-membrane lipid adduct. In this paper we present evidence that N-OH-AAF is deacetylated by a microsomal enzyme to form N-hydroxy-2-aminofluorene and this is then oxidized to 2-nitrosofluorene which adds covalently to membrane lipid double bonds to form N-O-LAF. Various antioxidants, peroxidase inhibitors, and P450 substrates and inhibitors were ineffective in altering the amount of N-O-LAF formed from N-OH-AAF; but two esterase inhibitors, dietyl-rho-nitrophenylphosphate and alpha-toluene-sulfonyl fluoride, prevented N-O-LAF formation. Of the following purified enzymes tested: porcine liver carboxyl esterase, pepsin, chymotrypsin, cathepsin D, ficin, papain, leucine aminopeptidase, Naja naja phospholipase, acetylcholinesterase (type I), trypsin (type I and V) and epoxide hydrase; only carboxyl esterase was effective in deacetylating N-OH-AAF.     

Interaction of RecA protein with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene and 4-hydroxyaminoquinoline 1-oxide.

Interaction of RecA protein of Escherichia coli with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and 4-hydroxyaminoquinoline 1-oxide (4HAQO) was investigated. RecA protein bound more efficiently to modified DNA than to unmodified DNA as judged by filter-binding and gel electrophoresis assay. The binding of RecA protein with modified DNA resulted in the stimulation of ATPase activity and the activation for RecA protein to stimulate the repressor cleavage. These abilities of RecA protein were increased proportionally to the number of adducts in the plasmid DNA (0-5 adducts). Apurinic and alkylated DNA did not activate RecA protein. We suggest that modification of DNA by N-OH-AAF and 4HAQO provides binding sites for RecA protein and may act as an activation signal for SOS response.     

Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures.

Sodium selenite (Na2SeO3) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetylaminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 X 10(-6) M Na2SeO3 resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na2SeO3 concentrations (7.90 X 10(-6) and 1.19 X 10(-5) M) resulted in a three-fold increase in the SCE frequency above background level (6--7 SCEs/cell). Exposure of lymphocytes to 1 X 10(-4) M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 +/- 0.75 while a similar exposure to 2.7 X 10(-5) M N-OH-AAF resulted in 13.61 +/- 0.43 SCEs/cell. Simultaneous addition of the high Na2SeO3 concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25--30% and 11--17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Species variation in bladder cell and liver cell activation of acetylaminofluorene

The metabolism and mutagenicity of 2-acetylaminofluorene were measured using freshly prepared intact bladder and liver cells from the cow, dog and rat. High pressure liquid chromatography was used to separate 2-acetylaminofluorene metabolites, andSalmonella typhimurium, strain TA98, was used to detect mutagenic intermediates. Species differences as well as animal-to-animal variation within a species were observed. Mutagenic activity with 2-acetylaminofuorene was greater with cow bladder cells than with dog or rat bladder cells. However, dog bladder cells were most active in metabolizing 2-acetylaminofluorene, and rat bladder cells were least active. Liver cells from all three species metabolized 2-acetylaminofluorene to mutagens forSalmonella, with dog and cow cells being more active than rat liver cells. However, cow liver cells were the most active in metabolizing 2-acetylaminofuorene, followed by rat and dog cells. With all cell types studied, except rat bladder cells, aminofluorene was the major metabolite detected. Carbon and N-hydroxylated products were produced by liver and bladder cells of the three species and glucuronide and sulfate conjugates of the metabolites were detected from both cell types. Correlations between mutagenic activity and the level of metabolism or any individual metabolite were not apparent. The data suggest that the relative contribution of bladder cell metabolism in aromatic amine induced bladder cancer may vary with the species.Abbreviations AAF 2-acetylaminofluorene - 4-ABP 4-aminobiphenyl - AF aminofluorene - BZ benzidine - cytochrome P-450 a collective term for all forms of the cytochrome P-450 polysubstrate mono-oxygenases - FMO flavin mono-oxygenases - HPLC high pressure liquid chromatography - MNNG N-methyl-N-nitro-N-nitrosoguani-dine - 2-NA 2-naphthylamine - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 5-OH-AAF 5-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8OH-AAF 8-hydroxy-2-acetylaminofluorene - 9-OH-AAF 9-hydroxy-2-acetylaminofluorene - UDS unscheduled DNA synthesis     

Modulation of genotoxic and cytotoxic effects of aromatic amines in monolayers of rat hepatocytes

Cultured rat hepatocytes exposed to 2-acetylaminofl uorene (AAF), 2-aminofl uorene (AF) or N-hydroxy-2-acetylaminofluorene (N-OH-AFF) for 3 hrs resulted in an increase in DNA repair measured as unscheduled DNA synthesis, with N-OH-AAF > AAF > AF. Cytotoxic effects were only seen with N-OH-AAF above 10^–6 M. -Naphthof avone increased the unscheduled DNA synthesis and cytotoxic effects of N-OH-AAF, whereas it decreased DNA repair and the covalent binding of AAF to cellular proteins. In contrast, very little effects of paraoxon were seen on the repair synthesis elicited by AAF, AF or N-OH-AAF. The addition of ascorbate reduced the covalent binding of AAF, the DNA repair synthesis caused by AAF and N-OH-AAF, and the cytotoxic effects of N-OH-AAF. The addition of pentachlorophenol or salicylamide all resulted in similar effects as ascorbate, through reduction of sulfation. Galactosamine, an inhibitor of glucuronidation, and the nucleophile GSH caused no or only minor effects of the activation of AAF, AF or N-OH-AAF as judged from the endpoints tested. These results are consistent with an arylnitrenium ion, a sulfate ester or a free radical as the arylamine metabolite causing cellular DNA damage, whereas the sulfate ester or a radical intermediate may be responsible for the cytotoxic effects of N-OH-AAF.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene - cytochrome P-450 a collective term for all forms of the cytochrome P-450 polysubstrate monooxygenase - DMSO dimethyl sulfoxide - HU hydroxyurea - S-9 9000 g supernatants - LDH lactate dehydrogenase - UDS unscheduled DNA synthesis - ANF -naphthoflavone - GSH glutathione - PCP pentachlorophenol - MET metyrapone - PAR paraoxon - DEM dimethylmaleate     

Main pathway for mutagenic activation of 2-acetylaminofluorene by guinea pig liver homogenates.

The activation pathway of 2-acetylaminofluorene (AAF) to N-hydroxy-2-amino-fluorene (N-OH-AF), a potent mutagen to $\text{Salmonella}$, by guinea pig liver postmitochondrial supernatant fraction (S-9 fraction) was studied. 2-Aminofluorene (AF), as well as N-hydroxy-2-acetylaminofluorene (N-OH-AAF, Takeishi et al., Mutation Res. in press), was detected as a metabolite of AAF. The mutagenicities of AF and N-OH-AAF comparable to that of AAF were inhibited by antiserum against NADPH-cytochrome $\text{c}$ reductase and by paraoxon, respectively. These data indicate that in the mutagenic activation of AAF, N-OH-AF can be produced by both N-hydroxylation of AF and deacetylation of N-OH-AAF. Furthermore, the data on the relative contribution of paraoxon-sensitive activation pathway to mutagenicities of AAF and N-OH-AAF led to a conclusion that deacetylation of AAF followed by N-hydroxylation to produce N-OH-AF is the main pathway for the mutagenic activation of AAF by guinea pig liver S-9 fraction.     

Bacterial mutagenesis and host cell DNA damage by chemical carcinogens in the Salmonella/hepatocyte system

Coincubation of isolated and intact rat hepatocytes and Salmonella typhimurium, (Salmonella/hepatocyte system) strain TA 98 was employed to determine both bacterial mutagenicity and DNA damage in the hepatocytes as measured by alkaline elution, following treatment with 2-acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Both the mutagenicity and the rate of DNA elution were dose-dependent for all three compounds. N-OH-AAF was 5 times more mutagenic and caused 80–100 times more DNA damage in the hepatocytes than AAF and AF when compared on a molar basis. The Salmonella/hepatocyte system may provide a more comprehensive evaluation of the potential genotoxic effect of chemicals than the currently used microbial mutagenesis sytems.     

Bioactivation of the hepatocarcinogen N-hydroxy-2-acetylaminofluorene by sulfation in the rat liver changes during the cell cycle.

Sulfation activity towards N-hydroxy-2-acetylaminofluorene and 4-nitrophenol was determined in male rat liver cytosol at several time points after partial hepatectomy corresponding to G1-, S-, and M-phase. N-hydroxy-2-acetylaminofluorene sulfation activity decreased by 80% when hepatocytes entered the G1-phase. This lower activity was maintained during the S-phase and M-phase, but was restored when hepatocytes entered the G0-phase again. Sulfation activity towards 4-nitrophenol did not alter after hepatectomy. Various other cytosolic enzyme activities were determined after hepatectomy to investigate the specificity of the decrease in sulfation activity. Lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities were increased in the S- and M-phase by maximally 80% and 60%, respectively. Glutathione-S-transferase and glutamate-pyruvate transaminase activity did not alter during the cell cycle. These results indicate that sulfation of N-hydroxy-2-acetylaminofluorene in hepatocytes may depend on the phase of the cell cycle. The relevance of the finding is discussed in relation to the resistance of proliferating (pre)neoplastic hepatocytes to the toxic and mitoinhibitory effects of N-hydroxy-2-acetylaminofluorene.     

32P-adduct assay: Short- and long-term persistence of 2-acetylaminofluorene-DNA adducts and other applications of the assay

³²P-Postlabeling techniques have been developed to detect and measure adducts formed by covalent binding of carcinogens of Known or unknown origin with DNA (and RNA). The assay is applicable to various classes of chemical carcinogens and permits detection of many adducts at attomole (10^–18 mol) level using microgram amounts of DNA. Here, we demonstrate the application of the assay for the analysis of short- and long-term persistence of 2-acetylaminofluorene-DNA adducts in rat liver in vivo and also outline examples illustrating the applicability of the procedure to different experimental problems.Abbreviations AAF 2-acetylaminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene     

Biochemical changes after hepatic injury by allyl alcohol and N-hydroxy-2-acetylaminofluorene.

Administration of hepatotoxic doses of allyl alcohol and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) TO adult male rats produced periportal necrosis and functional derangement of the hepatic endoplasmic reticulum within 24 h. The rates of N-demethylation of ethylmorphine and p-hydroxylation of aniline were decreased 6 h following allyl alcohol administration, but cytochromes P-450 and b5 were unchanged. In contrast, administration of NOH-AAF decreased cytochromes P-450 and b5 and the rate of aniline p-hydroxylation, but did not change the rate of N-demethylation of ethylmorphine or the activities of cytochrome c reductase and glucose-6-phosphatase. No decrease was observed in the activity of the cytosol enzyme, DT diaphorase, following allyl alcohol treatment. The changes by these periportal hepatotoxins were compared with those produced both by central and midzonal hepatotoxins and with changes occurring in the liver after surgical partial hepatectomy.     

Free radical activation of N-hydroxy-2-acetylaminofluorene by methemoglobin and hydrogen peroxide

We have shown that N-hydroxy-2-acetylaminofluorene, a metabolite of 2-acetylaminofluorene, is converted via a nitroxide free radical into N-acetylaminofluorene and 2-nitrosofluorene by H2O2 in the presence of methemoglobin. Utilizing optical methods, we have demonstrated that the rate of 2-nitrosofluorene production parallels that of N-hydroxy-2-acetylaminofluorene oxidation. This evidence is consistent with a model whereby two molecules of N-hydroxy-2-acetylaminofluorene yield two nitroxide free radicals which then dismutate to form one molecule of N-acetoxy-2-acetylaminofluorene and one molecule of 2-nitrosofluorene. The Km of N-hydroxy-2-acetylaminofluorene for this reaction is 114 microM with a Vmax of 50 microM/min.     

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.     

Inhibition of 4-nitroquinoline 1-oxide induced unscheduled DNA synthesis in primary cultures of rat urothelial cells by dicumarol and pyrophosphate

Primary cultures of rat urothelial cells were exposed to hydroxyurea, [3H]thymidine, and 4-nitroquinoline 1-oxide (NQO) or N-hydroxy-4-aminoquinoline 1-oxide (HAQO) in a serum-free media for 2 h; unscheduled DNA synthesis (UDS) was measured by autoradiography. Both NQO and HAQO produced unscheduled DNA synthesis. Dicumarol, an inhibitor of NQO nitroreductase, inhibited the activity of NQO and, to a lesser extent, HAQO. Pyrophosphate, an inhibitor of seryl-AMP synthetase, inhibited the activity of both compounds. Neither dicumarol nor pyrophosphate, under similar experimental conditions, inhibited the activity of N-hydroxy-N-2-acetylaminofluorene (N-OH-AAF). These results support the idea that nitro-reductase and seryl-AMP synthetase may be involved in the activation of NQO.     

Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells

Wild-type Chinese hamster ovary cells (AA8) and five excision-deficient clones derived from the AA8 line (UV-4, UV-5, UV-20, UV-24 and UV-41) were exposed to ultraviolet light and then analyzed for their ability to incorporate [3H]thymidine and to initiate as well as elongate replicon-sized DNA fragments. After exposure to ultraviolet light, all cell lines exhibited a depression in the rate of thymidine incorporation. For exposures of 4.0 J/m2 or higher the wild-type cells recovered normal rates of thymidine incorporation within a few hours, while none of the excision-deficient lines exhibited complete recovery. For fluences below 4.0 J/m2 all but the UV-5 line exhibited at least some recovery. The ability to elongate DNA chains appeared to correlate with the thymidine incorporation data, with the UV-5 line exhibiting the strongest blockage of DNA chain elongation, the AA8 line exhibiting the least blockage, and the UV-20 line exhibiting an intermediate response. All cell lines exhibited a decrease in the distance between replication origins, thus supporting models which propose that exposure to ultraviolet light results in the use of alternative sites for the initiation of replication.     

Effects of harman and norharman on the metabolism and genotoxicity of 2-acetylaminofluorene in cultured rat hepatocytes

Monolayers of rat hepatocytes metabolize 0.25 m M 2-acetylaminofluorene (AAF) to various ether-extractable, water-soluble as well as covalently bound products. The major ether-extractable metabolite formed is 2-aminofuorene (AF), followed by 7-OH-AAF and 9-OH-AAF. Pretreatment of rats with the inducer Aroclor 1254 (PCB) increased the metabolism of AAF and caused an increased DNA repair synthesis in hepatocytes exposed to AAF or AF. With N-OH-AAF, a decreased genotoxic response in PCB-treated cells compared to control cells was seen. The addition of harman and norharman decreased the metabolism of AAF to ether-extractable metabolites, water-soluble metabolites and metabolites covalently bound to macromolecules. In contrast, the DNA-repair synthesis caused by the same concentrations of AAF was increased by harman. One explanation for this apparent discrepancy could be that the aromatic amines changed the metabolism of harman and norharman in such a way that these compounds were converted into genotoxic metabolites.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-ydroxy-2-acetylaminofluorene - PCB polychlorinated biphenyls, Aroclor 1254 - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin - TdR thymidine - Trp-P-1 3-amino-1,4dimethyl-5H-pyrido(4,3b)indole - Trp-P-2 3-amino-l-methyl-5H-pyrido(4,3b)indole - UDS unscheduled DNA synthesis     

The binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated rat liver nuclei: effects of glutathione and methionine

The covalent binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated, intact rat liver nuclei was studied. The chemically synthesized 2-acetylaminofluorene-N-sulfate became covalently bound to DNA and protein to form adducts, 50% to 60% of which retained the N-acetyl group. Glutathione decreased the covalent binding of acetylated adducts to DNA by 18% and to protein by 50%. Methionine was more effective; it decreased DNA binding by 52% and protein binding by 79%. N-Hydroxy-2-acetylaminofluorene was deacetylated by the nuclear preparation. Almost exclusively, deacetylated 2-aminofluorene adducts to DNA and protein were formed. Glutathione decreased the covalent binding of deacetylated adducts to DNA by only 14%. Protein binding, however, was decreased by 57%. Methionine had no effect on the formation of these adducts to DNA and protein. Formation of 2-aminofluorene-glutathione conjugates was reduced by ascorbic acid by 65%. Covalent binding of deacetylated adducts to DNA and protein, however, was not decreased by ascorbic acid. These data suggest that "harder" nucleophiles like methionine can be used to protect macromolecules in vivo from damage by "hard" electrophiles such as those generated from the reactive 2-acetylaminofluorene-N-sulfate. However, such nucleophiles seem not to be effective with N-hydroxylamines, such as N-hydroxy-2-aminofluorene, formed by deacetylation of N-hydroxy-2-acetylaminofluorene.     

Nucleotide excision repair functions in the removal of chromium-induced DNA damage in mammalian cells

Some hexavalent chromium (Cr(VI))-containing compounds are human lung carcinogens. While ample information is available on the genetic lesions produced by Cr, surprisingly little is known regarding the cellular mechanisms involved in the removal of Cr-DNA adducts. Nucleotide excision repair (NER) is a highly versatile pathway that is responsive to a variety of DNA helix-distorting lesions. Binary Cr-DNA monoadducts do not produce a significant degree of helical distortion. However, these lesions are unstable due to the propensity of Cr(III) to form DNA adducts (DNA interstrand crosslinks, DNA-protein/amino acid ternary adducts) which may serve as substrates for NER. Therefore, the focus of this study was to determine the role of NER in the processing of Cr-DNA damage using normal (CHO-AA8) and NER-deficient [UV-5 (XP-D); UV-41 (ERCC4/XP-F)] hamster cells. We found that both UV-5 and UV-41 cells exhibited an increased sensitivity towards Cr(VI)-induced clonogenic lethality relative to AA8 cells and were completely deficient in the removal of Cr-DNA adducts. In contrast, repair-complemented UV-5 (expressing hamster XPD) and UV-41 (expressing human ERCC4) cells exhibited similar clonogenic survival and removed Cr-DNA adducts to a similar extent as AA8 cells. In order to extend these findings to the molecular level, we examined the ability of Cr(III)-damaged DNA to induce DNA repair synthesis in cell extracts. Repair synthesis was observed in reactions using extracts derived from AA8, or repair-complemented, but not NER-deficient cells. Cr(III)-induced repair resynthesis was sensitive to inhibition by the DNA polymerase δ/ε inhibitor, aphidicolin, but not 2′,3′-dideoxythymidine triphosphate (ddTTP), a polymerase β inhibitor. These results collectively suggest that NER functions in the protection of cells from Cr(VI) lethality and is essential for the removal of Cr(III)-DNA adducts. Consequently, NER may represent an important mechanism for preventing Cr(VI)-induced mutagenesis and neoplastic transformation.