Susceptibility to Aflatoxin B₁-Induced Carcinogenesis Correlates with Tissue-Specific Differences in DNA Repair Activity in Mouse and in Rat

Introduction

Aflatoxin B₁ (AFB₁) is a member of a family of difuranocoumarins produced by Aspergillus flavus and related fungi (1). AFB₁ is the most hepatotoxic and carcinogenic of the aflatoxins, and is an established risk factor for hepatocarcinogenesis in humans (2). The lung can also be a target of AFB₁, following dietary or inhalational exposure (3–6) . AFB₁ is biotransformed by cytochromes P450 (7), lipoxygenase (8, 9) , and prostaglandin H synthase (8, 9) to the highly reactive AFB₁-8,9-exo-epoxide, which binds preferentially to the N⁷ position of guanine (Gua) residues in DNA, forming AFB₁-N⁷-Gua (10, 11) . AFB₁-N⁷-Gua can hydrolyze spontaneously to the more chemically stable and biologically persistent formamidopyrimidine adduct (AFB₁-FAPY; ref. 12). Both AFB₁-N⁷-Gua and AFB₁-FAPY adducts result in mostly G to T transversions at the site of the lesion and cause some mutations 5′ to the lesion (13), consistent with the principal mutation in human and rodent liver (14, 15) and lung tumors (9) attributed to AFB₁ exposure.

Susceptibility to the toxic and carcinogenic effects of AFB₁ varies markedly between species (16). The rat, duck, and trout are highly susceptible to AFB₁-induced hepatocarcinogenesis, whereas the monkey, mouse, and hamster are relatively resistant (1). Although mice are resistant to AFB₁-induced hepatocarcinogenesis, they are susceptible to pulmonary tumorigenesis (9, 17) . Mechanisms underlying tissue- and species-related differences in susceptibility to the carcinogenic effects of AFB₁ are not well established, however, evidence has accumulated supporting the importance of biotransformation. The difference in AFB₁ hepatocarcinogenicity between rats and mice has been attributed to the expression of an α-class glutathione-S-transferase in mouse liver capable of efficient enzymatic conjugation of AFB₁-exo-epoxide with glutathione (18, 19) .

Although bioactivation and consequent DNA damage are critical determinants of chemical mutagenicity, the maintenance of genetic integrity depends on the ability to repair damaged DNA. The importance of nucleotide excision repair (NER) in the removal of AFB₁-induced DNA damage is well established (20–22) . Although more than 40 proteins are involved in NER, the process consists of two main steps: (a) the incision reaction, including damage recognition, asymmetric incision of the lesion-containing DNA strand on both sides of the lesion, and excision of the damaged oligonucleotide; and (b) gap filling by DNA polymerization and ligation (23). The process of NER has been reproduced in a biochemical assay in vitro (24, 25) , in which repair activity of cell-free nuclear protein extracts prepared from either whole tissue or cell lines is measured by the extent of DNA repair synthesis in damaged plasmid DNA, detected as incorporation of radiolabeled nucleotides. In vitro assessment of NER on naked plasmid DNA resembles genomic repair in vivo (25), and defective repair activity has been observed in extracts from repair-deficient xeroderma pigmentosum cells (26, 27) .

In the present study, we investigated whether the DNA repair activity of extracts from mouse lung and liver and rat liver toward plasmid DNA with a known number of AFB₁-N⁷-Gua or AFB₁-FAPY adducts correlated with species- and tissue-specific susceptibility to AFB₁-induced carcinogenesis. We also investigated whether in vivo treatment of mice and rats with a tumorigenic dose of AFB₁ had any effect on in vitro repair activity.

Materials and Methods

Animal Treatments

Male Sprague-Dawley rats (345-355 g, Charles River Canada, Inc., St. Constant, PQ, Canada) and female CD-1 mice (25-30 g, Charles River) were housed with a 12-hour light/dark cycle and provided food and water ad libitum. These strains of rats and mice were chosen because previous demonstration of interspecies differences in susceptibility to AFB₁ was conducted in them, and they have been used for mechanistic studies (18, 19) . Mice were treated with vehicle (50 μL DMSO, i.p.) or with 50 mg/kg AFB₁ (Sigma Corp., St. Louis, MO; 50 μL in DMSO, i.p.), a dose that has been shown to result in pronounced pulmonary tumorigenesis, but not hepatocarcinogenicity, in the AC3F1 mouse (17). In an independent experiment, mice were treated with 0.25 mg/kg AFB₁ (50 μL in DMSO, i.p.) and rats were treated with vehicle (300 μL DMSO, i.p.), or 0.25 mg/kg AFB₁ (300 μL in DMSO, i.p.), a dose that does not produce overt hepatotoxicity but is sufficient to cause neoplastic liver changes in rats if given daily for 2 weeks (28). Two hours following AFB₁ administration, when covalent binding to DNA is maximal (19, 29, 30) , mice and rats were killed by cervical dislocation or decapitation, respectively. Lungs and livers were perfused with 10 mmol/L Tris and 1 mmol/L EDTA (pH 7.9), excised, finely chopped, snap-frozen, and stored at −80°C until required for extract preparation.

Cell-Free Whole Tissue Nuclear Protein Extract Preparation

Nuclear protein extracts from ∼1.5 to 1.8 g of tissue were prepared from the livers of individual rats or mice and from the lungs of pooled groups of four mice each. Tissue pieces frozen in liquid nitrogen were pulverized to a fine powder with a mortar and pestle. Cell-free tissue nuclear protein extracts active in DNA repair synthesis were prepared as described (24, 26) with the following modifications: (a) hypotonic lysis of tissue was done for 30 minutes under gentle rotation; (b) dialysis was done against three changes of 200 mL (per Slide-A-Lyzer cassette, Pierce Biotechnology Inc., Rockford, IL) of buffer for 18 hours, and changed after 1 and 5 hours of dialysis. Protein content of extracts was determined by the method of Lowry et al. (31). Extracts from mouse lung and liver and rat liver typically contained 11, 28, and 20 mg protein, respectively. Extracts were snap-frozen in liquid nitrogen and stored at −80°C.

Preparation and Analysis of AFB₁-Adducted DNA

AFB₁-8,9-exo-epoxide was prepared by oxidation of AFB₁ by dimethyldioxirane (32), recrystallized from 1:2 (v/v) acetone/dichloromethane, characterized by ¹H-nuclear magnetic resonance (400 MHz, Brüker Instruments), stored under nitrogen at −20°C, and dissolved in anhydrous acetone prior to use. AFB₁-N⁷-Gua- and AFB₁-FAPY-adducted calf thymus DNA (sodium salt, Sigma) were prepared as described (33) and acid hydrolysates of the adducted DNA subjected to isocratic reversed phase high-performance liquid chromatography (LCDB18 C₁₈ column, 250 × 4.6 mm, Supelco, St. Louis, MO) as previously described (34). AFB₁-DNA adducts were detected at 365 nm using a Waters Lambda-Max LC spectrophotometer (Model 481).

A 3875 bp plasmid derived from pBluescript (Stratagene, La Jolla, CA) was grown in E. coli XL-1 Blue in LB broth and isolated using a Qiagen Plasmid Giga Kit (Qiagen, Valencia, CA). AFB₁-N⁷-Gua and AFB₁-FAPY adducted plasmid DNA were prepared as described (35) with a minor change; AFB₁-adducted plasmid DNA was purified by sodium acetate/isopropanol precipitation.

Immunoprecipitation of Proliferating Cell Nuclear Antigen in Mouse Liver Nuclear Protein Extracts

Mouse liver nuclear protein extract (2.5 mg) was precleared with protein G Sepharose beads (Amersham Biosciences Corp., Piscataway, NJ) for 1 hour at 4°C prior to immunoprecipitation. Precleared extracts were incubated overnight at 4°C either in the presence or absence of 7 μg of α-human proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody (PC10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by incubation with protein G Sepharose beads for 5 hours at 4°C. Supernatants were retrieved and stored at −80°C for evaluation of repair activity. Supernatants and proteins recovered from beads were subjected to SDS-PAGE and immunoblotting was achieved according to the manufacturer's recommendations.

In vitro Repair Reactions

Repair Synthesis Assay. The repair synthesis assay (24, 36) was done in a total volume of 50 μL containing either 400 ng of AFB₁-adducted plasmid DNA or undamaged DNA, 40 mmol/L HEPES-KOH (pH 7.8), 0.5 mmol/L DTT, 4.0 μmol/L dTTP, 20 μmol/L of each dGTP, dCTP and dATP, 23 mmol/L phosphocreatine, 18 μg bovine serum albumin (nuclease-free, Sigma), 2.5 μg creatine phosphokinase, 2.0 mmol/L ATP, 5.0 mmol/L MgCl₂, 0.4 mmol/L EDTA, 100 mmol/L KCl, 120 μg tissue protein extract and 2.0 μCi [α³²P] dTTP (Amersham). Samples were incubated for 3 hours at 30°C. Repair reaction was terminated by adjusting the concentration of EDTA to 20 mmol/L. Following treatment with 7.0 mg/mL RNase (37°C for 10 minutes) and with 350 μg/mL proteinase K (with 0.5% SDS at 65°C for 30 minutes), plasmid DNA was purified by extraction with phenol/chloroform/isoamyl alcohol (25:24:1; v/v/v), isopropanol precipitated, washed in 70% ethanol, dried and linearized with 1 unit of EcoR1 (New England Biolabs, Beverly, MA). The restriction digestion was terminated by addition of DNA gel loading solution (0.02% bromophenol blue, 6.0 mmol/L EDTA, 6.0% glycerol). Electrophoresis was done through a 1% agarose gel with 0.5 μg/mL ethidium bromide and with 40 mmol/L Tris base/40 mmol/L acetic acid/1 mmol/L EDTA as the tank buffer. Repair synthesis assay was done in duplicate for each nuclear protein extract.

Incision Assay. The incision assay was done using either 400 ng of UV irradiated plasmid DNA or undamaged DNA as previously described (36, 37) . In the incision reaction, repair synthesis is prevented by limiting the nucleotide pool and by inhibiting DNA polymerase activity in protein extracts with aphidicolin. Incisions are then quantitatively detected by [α³²P]dTTP incorporation catalyzed by the Klenow fragment of E. coli DNA polymerase I at nicked sites in plasmids purified from incision reactions. The incision assay was modified as follows: (a) following the incision reaction, extracted DNA was redissolved in TE buffer [10 mmol/L Tris, 1 mmol/L EDTA (pH 7.9)] by incubation at 30°C for 15 minutes, (b) the repair synthesis step catalyzed by E. coli DNA polymerase I large fragment (New England Biolabs) was done at 37°C for 10 minutes using 20 μmol/L of each dGTP, dCTP, and dATP, 4.0 μmol/L dTTP, and 2.0 μCi [α³²P]dTTP. Incision assay was done in duplicate for each nuclear protein extract.

Quantification of Repair and Data Analysis. Normalization for plasmid DNA recovery was done by densitometry of a photograph of the gel (ChemImager 4000, Alpha Innotech Corporation, San Leandro, CA). Incorporation of [α³²P]dTTP was determined by phosphor imaging (Molecular Dynamics, Piscataway, NJ) of the dried gel. Statistically significant differences in repair activity between tissue type or treatment groups and type of adduct were determined by two-way repeated measures ANOVA followed by Bonferroni correction method (GraphPad Prism 4 software). Homogeneity of variance was verified by Cochran's test prior to conducting the ANOVA. P < 0.05 was considered significant in all cases.

Results

Preparation and Characterization of AFB₁-Adducted Plasmid DNA Substrate for Repair. AFB₁-8,9-exo-epoxide was recovered as a solid product with a ratio of 25:1 exo/endo as determined by ¹H-nuclear magnetic resonance using the integration for H8 of the AFB₁-8,9-epoxide (38). Upon the reaction of AFB₁-exo-epoxide with calf thymus DNA, AFB₁-N⁷-Gua represented 98% of formed adducts, and two forms of AFB₁-FAPY were identified (∼1% AFB₁-FAPY major, <1% AFB₁-FAPY minor; ref. 12). Treatment of AFB₁-adducted DNA with sodium hydroxide resulted in the complete conversion of AFB₁-N⁷-Gua to AFB₁-FAPY major (96%) and AFB₁-FAPY minor (4%). AFB₁-DNA adduct formation was dependent on the concentration of AFB₁-8,9-exo-epoxide with both calf thymus and plasmid DNA (data not shown), and using this relationship, substrates with 10 or 100 adducts per plasmid of either AFB₁-N⁷-Gua or AFB₁-FAPY adducts were prepared. These DNA substrates were found to be stable under the experimental conditions used for the repair synthesis assay (see above). No nicks or relaxation of the adducted plasmids were observed by gel electrophoresis of the DNA substrates after incubation at 30°C for 3 hours. During the 3-hour incubation of AFB₁-N⁷-Gua-adducted plasmid, 3.0% AFB₁-N⁷-Gua was converted to AFB₁-FAPY major and 2.0% AFB₁-N⁷-Gua was released from DNA, presumably forming apurinic sites. No increase in the amount of AFB₁-diol was observed, indicating that hydrolysis of AFB₁-N⁷-Gua was negligible under these experimental conditions. The AFB₁-FAPY-adducted substrate initially consisted of 96% AFB₁-FAPY major and 4% AFB₁-FAPY minor and was unchanged by incubation for 3 hours at 30°C.

DNA Repair Synthesis Assay Validation for AFB₁-Adducted DNA Substrates. Although repair synthesis was detectable in undamaged DNA, activity was higher towards AFB₁-N⁷-Gua-adducted substrate with 10 adducts per plasmid for all tissues ( Table 1 ). In contrast to mouse and rat liver, mouse lung could not catalyze repair synthesis of AFB₁-N⁷-Gua at 100 adducts per plasmid; therefore, AFB₁-N⁷-Gua-adducted substrate with 10 adducts per plasmid was used for all other determinations.

Repair of AFB₁-N⁷-Gua and AFB₁-FAPY was decreased by 29% to 100% in mouse liver nuclear protein extracts immunodepleted for PCNA, consistent with NER being the major pathway of AFB₁-induced DNA damage (20–22) . The effectiveness of immunodepletion was confirmed by immunoblotting (n = 2; Fig. 1 ). Repair activity of mouse liver extracts was nondetectable (<0.3 amol [α³²P]dTTP incorporated per microgram of DNA) in the absence of ATP and was decreased by 72% when creatine phosphokinase was omitted (n = 1, data not shown).

Repair of AFB₁ DNA Damage by Mouse Lung and Liver and Rat Liver Nuclear Protein Extracts. Damage-specific repair activity was nondetectable with <80 μg of mouse lung nuclear protein extract. Due to the low yield and concentration of protein extracted from mouse lung, it was not possible to include more than 120 μg of protein per sample. To compare the repair activity between tissue extracts, the amount of protein was adjusted to 120 μg for all experiments. Mouse liver exhibited ∼5- and 30-fold greater repair activity towards AFB₁-N⁷-Gua and AFB₁-FAPY, respectively, than did lung ( Fig. 2 ). Furthermore, mouse liver repair activities were ∼6- and 4-fold higher than those of rat liver towards AFB₁-N⁷-Gua and AFB₁-FAPY ( Fig. 2). Mouse liver and lung exhibited greater repair activity towards AFB₁-N⁷-Gua than towards AFB₁-FAPY.

Effect of in vivo Treatment of Mice with 50 mg/kg AFB₁ on Repair Synthesis and Incision Activities in Liver and Lung. Repair synthesis activity towards AFB₁-N⁷-Gua was inhibited by 85% in mouse lung following in vivo treatment with 50 mg/kg AFB₁ 2 hours prior to tissue harvesting ( Fig. 3A ). AFB₁-N⁷-Gua damage-specific incision activity was lower than that measured towards undamaged DNA, presumably due to the inhibition of the Klenow fragment by AFB₁-DNA adducts (refs. 39–41 and data not shown). We therefore determined the incision activity of mouse lung extracts using UV-damaged plasmid DNA as a substrate. Lung extracts from vehicle-treated mice showed UV damage–specific incision activity of 4,130 ± 1,980 amol [α³²P]dTTP incorporated per microgram of DNA, incising UV damage ∼77-fold more actively than lung extracts from AFB₁-treated mice (53.7 ± 93.0 amol [α³²P]dTTP incorporated per microgram of DNA; n = 3, P < 0.05). Lung extracts from vehicle-treated mice showed UV-damage repair synthesis activity of 3,940 ± 2,000 amol [α³²P]dTTP incorporated per microgram of DNA, whereas repair synthesis activity towards UV damage was inhibited by 82% in lung extracts from AFB₁-treated mice (706 ± 1,220 amol [α³²P]dTTP incorporated microgram of DNA; n = 3, P < 0.05). In contrast, repair activity towards AFB₁-N⁷-Gua was ∼3.5-fold higher in liver extracts from AFB₁-treated mice compared with control, but was nondetectable towards AFB₁-FAPY ( Fig. 3B).

Effect of In vivo Treatment of Mice and Rats with 0.25 mg/kg AFB₁ on Repair Synthesis in Liver. Although the trend seemed to be that in vivo treatment of mice and rats with 0.25 mg/kg AFB₁ increased repair synthesis activity towards AFB₁-N⁷-Gua in mouse liver and decreased repair synthesis activity in rat liver ( Fig. 4 ), this effect did not attain statistical significance (P > 0.05).

Discussion

Although AFB₁ is an established hepatocarcinogen in humans and in several experimental animals, the carcinogenic response to AFB₁ varies markedly between species. Detoxification of AFB₁-8,9-exo-epoxide via conjugation with glutathione is believed to be a major determinant of species susceptibility to AFB₁-induced carcinogenesis (9, 18) . However, the results of this study support that differences in DNA repair are also likely to be important contributing factors to both the tissue- and species-specific susceptibility to AFB₁-induced carcinogenesis. Assuming that the ratio of protein involved in DNA repair to total nuclear protein extracted is similar for the two species, we have observed that rat liver has lower DNA repair activity than mouse liver, correlating with the species difference in susceptibility to AFB₁-induced hepatocarcinogenesis. Although data for mouse lung are not available, our results are consistent with previous studies which have shown that mouse liver has only 1.2% of the AFB₁-DNA adducts measured in rat liver 2 hours following in vivo treatment with 0.25 mg/kg AFB₁ (19). We also suggest that the susceptibility of mouse lung relative to liver to AFB₁ carcinogenicity is in part attributable to: (a) lung having much lower DNA repair activity than liver, (b) inhibition of repair in lung by AFB₁, and (c) induction of repair in liver by AFB₁. The inability of mouse lung to catalyze repair of AFB₁-N⁷-Gua at 100 adducts per plasmid might conceivably contribute to the higher susceptibility of mouse lung to AFB₁ carcinogenesis relative to liver, and could be due to differential sensitivity of mouse lung DNA polymerases to inhibition by AFB₁-DNA adducts. Although in vitro assessment of NER on naked plasmid DNA with 10 adducts per molecule resembles genomic repair in vivo (24–26) , this may not hold true for higher numbers of adducts.

Repair activity towards AFB₁-N⁷-Gua was increased in liver, but not in lung, following in vivo treatment with 50 mg/kg AFB₁ at a time point (2 hours) when the levels of AFB₁-DNA adducts are maximal (29), and when AFB₁-N⁷-Gua represents ∼83% of adducts (42). Induction of repair in liver was also AFB₁ dose–related because repair activity was not increased by treatment of mice with 0.25 mg/kg AFB₁. Until recently, it was commonly accepted that mammalian excision repair occurs constitutively. Furthermore, the existence of the well characterized bacterial SOS response, wherein the DNA repair system requires induction by damage, has not been shown in eukaryotic cells. However, there are reports suggesting that UV irradiation can induce NER in mammalian cells, increasing repair activity (43–45) . In particular, nuclear protein extracts from UV-irradiated RKO cells exhibited greater repair activity towards UV damage than extracts prepared from nonirradiated cells (43). Our results support the hypothesis that mammalian NER is inducible, not only in response to UV irradiation, but also following treatment with AFB₁, and this may also occur following treatment with other chemical carcinogens. Further investigation is required to elucidate the mechanism by which AFB₁ can induce repair activity in mouse liver. In the mouse, in addition to causing DNA damage, AFB₁ can modulate repair activity in target tissues, influencing the susceptibility to AFB₁-induced carcinogenesis. The effect of AFB₁ on repair activity is a novel finding, and may prove to be an important mechanism underlying interspecies differences in susceptibility to other carcinogens.

Incision activity towards UV damage is inhibited in mouse lung following in vivo treatment with AFB₁; however, the mechanism of inhibition remains to be elucidated. Based on reports by others (46, 47) of selective inhibition of rat liver RNA polymerase II at 2 hours following in vivo treatment with 3 mg/kg AFB₁, and on the results of this study, we hypothesize that repair proteins or other proteins that regulate NER, specifically incision, the rate-limiting step of NER, are inactivated or down-regulated following in vivo administration of AFB₁.

The adduct referred to as AFB₁-FAPY is actually an equilibrium mixture of two rotameric forms (AFB₁-FAPY major and minor) that are separable by chromatography, as observed in this study and as reported by others (12). An experimental method for the exclusive synthesis of either form of AFB₁-FAPY adducts on DNA has not been reported and would be complicated by the conversion of AFB₁-FAPY minor, whose precise chemical structure remains elusive, to the major form (48). Whether AFB₁-FAPY major and AFB₁-FAPY minor are repaired with different efficiencies is unknown, so for the purposes of this study, we have considered the repair of AFB₁-FAPY to include both rotameric forms.

The differences in chemical structure of AFB₁-N⁷-Gua and AFB₁-FAPY cause these adducts to alter the structure of DNA differently (49). AFB₁-FAPY is less distortive to DNA architecture than is AFB₁-N⁷-Gua, making AFB₁-FAPY relatively resistant to repair (48); this is consistent with the observed persistence of AFB₁-FAPY in vivo and with results of the present study, in which nuclear extracts from mouse lung and liver repaired AFB₁-FAPY less effectively than AFB₁-N⁷-Gua. Although evidence exists suggesting that enzymatic removal of AFB₁-FAPY occurs by NER (50), bacterial FAPY-glycosylase has been shown to efficiently incise AFB₁-FAPY lesions in vitro (35, 51) . FAPY-glycosylase is expressed in rodent cells (52), but the relevance of this pathway in mammalian repair has not been established. Our observation that the repair of AFB₁-FAPY depends on PCNA supports the importance of NER. Interestingly, the differential effect of treatment of mice with 50 mg/kg AFB₁ on hepatic repair activity, i.e., induction of repair towards AFB₁-N⁷-Gua versus inhibition of repair towards AFB₁-FAPY, suggests that these two adducts may be repaired by different pathways.

In vivo NER can be divided into two pathways: (a) global genome repair (GGR), which is slow and repairs lesions throughout the entire genome, and (b) transcription-coupled repair (TCR), a preferential and highly efficient repair pathway for lesions on the transcribed strand of active genes (53). The enzymatic steps of TCR have not yet been reconstituted in vitro (54), but in vitro NER, as studied in this paper, has characteristics that mimic in vivo GGR (25). Although UV light–induced lesions, platinated DNA and psoralen adducts have been used extensively as substrates for in vitro studies of NER (24, 26) , this is the first study to investigate repair activities of nuclear protein extracts using AFB₁-N⁷-Gua- and AFB₁-FAPY-adducted DNA as substrates. NER efficiency is strongly dependent on the type of DNA lesion (54), emphasizing the importance of using AFB₁-DNA damage as a substrate for repair of relevance to AFB₁ toxicity. The determination of repair activity of tissue extracts is often problematic due to contamination by nonspecific nucleases which randomly nick undamaged DNA (36), resulting in high and variable background repair synthesis in undamaged DNA. In the present study, this disadvantage was overcome by small modifications of the procedure for extract preparation and by using a KCl concentration (100 mmol/L) known to minimize the activity of nonspecific nucleases (36). DNA repair synthesis of AFB₁-DNA damage by tissue extracts was dependent on the presence of PCNA, ATP, and an ATP regenerating system, consistent with NER being the major repair pathway for AFB₁-induced DNA damage (20–22) . PCNA is required for the repair synthesis step of NER (55) by binding to dually incised DNA and recruiting polymerases for DNA elongation. Furthermore, evidence for the role of PCNA in the incision and excision steps of NER is mounting but remains controversial (55). Although damage recognition is ATP-independent, ATP is required for the binding of all NER factors that participate in the incision and excision reactions (56).

TCR activity of tissue extracts is not addressed in this study, but mutations in nontranscribed genes or on the nontranscribed strand of active genes (repaired by GGR) are a better predictive marker for carcinogen-induced tumorigenesis than mutations in actively transcribed genes (53). The importance of GGR and a smaller role for TCR in avoiding genomic instability and cancer are exemplified by two inherited defects in NER in humans. Xeroderma pigmentosum patients, deficient in GGR, are much more susceptible to cancer in comparison to Cockayne syndrome patients, who have impaired TCR but normal GGR. TCR is crucial for cell survival and the lower cancer incidence in Cockayne syndrome patients is thought to be due to selective elimination of cells by apoptosis as a result of prolonged transcription blockage (53).

The results of this study show that species- and tissue-related differences in DNA repair activity correlate with susceptibility to AFB₁-induced carcinogenesis. Furthermore, in vivo treatment of mice with a tumorigenic dose of AFB₁ leads to a tissue-specific change in DNA repair activity that also correlates with susceptibility.