Transient structural change in hepatic DNA of rats chronically exposed to dimethylnitrosamine

Structural analysis by chromatography on benzoylated-DEAE-cellulose (BD-cellulose) has been made of hepatic DNA from rats treated for up to 21 weeks with dimethylnitrosamine (DMN). The carcinogen (1 mg/kg body wt) was administered on a daily basis by intraperitoneal injection. By comparison with preparations from saline-treated controls, the proportion of DNA retained by benzoylated-DEAE-cellulose in the presence of 1.0 M NaCl was increased by administration of the carcinogen. Variation in the result, dependent upon the time after the final dose, suggested a complex relationship between structural damage to DNA and duration of treatment. Structural damage was confirmed by S1 nuclease digestion. The observations imply that in the course of chronic administration, increasing time is required to complete DNA repair processes operative in rat liver.     

Target tissue DNA damage in inbred mouse strains with different susceptibility to the colon carcinogen 1, 2-dimethylhydrazine

We have compared liver, kidney and colon DNA damage, as singlestrand breaks, in mice with different strain-dependent susceptibilityto the colon-specific carcinogen 1, 2-dimethylhydrazine (DMH).The mouse strains studied were: AKR/J, DBA2 totally resistant;CD1, C57BL/6N moderately susceptible; SWR/J very susceptibleto DMH-induced carcinogenesis. DNA breaks were estimated fromthe elution rate constant (K) according to the alkaline elutiontechnique. At 4 h after carcinogen administration a substantialand comparable DNA damage was found in liver and kidney in allthe strains examined. The DNA fragmentation index, however,reached a maximum value at 2 h after treatment in the liverof the most susceptible strain (SWR/J). About 50% of the liverDNA damage detected in all five strains 4 h after DMH administrationpersisted at 24 h after treatment and was totally repaired at72 h. Kidney DNA damage decreased in 48 h toward the range ofcontrol values. In colon epithelial cells (the carcinogen targettissue) 2 and 4 h after DMH administration the amount of DNAsingle strand breaks was correletable with the strain sensitivityto the carcinogen. In the time interval studied (2–72h after DMH administration) the decrease of colon DNA damagewas linear in the resistant strains. In contrast, in the moresusceptible strain (SWR/J), the amount of DNA breaks remainedhigh up to 24 h after treatment and returned to background levelat 72 h.     

DNA polymerases in replication and repair of DNA during carcinogenesis induced by feeding N-acetylaminofluorene

To study the roles of DNA polymerases and ß duringreplication and repair of damaged DNA, use was made of the factthat during chronic treatment with carcinogens, replicationand repair do not necessarily follow the same time sequence.Early cell damage and restorative hyperplasia cause a transientwave of DNA synthesis, while repair replication might be expectedto continue throughout the period of treatment with the carcinogen.N-acetylaminoflluorene (AAF) was fed in the diet for periodsof up to 35 weeks, and at intervals during the feeding periodmeasurements were made of DNA synthesis in vivo, and off DNApolymerases a and ß as assayed in vitro after fractionation.The activity of polyararise increased and decreased with thetransient early wave of DNA synthesis. Polymerase ßshowed an initial rapid increase in activity which peaked beforethe increase in DNA synthesis, and then decreased. The decreasein activity may be due to the fact that, although AAF continuesto be fed in the diet, the foci and nodules which develop nolonger metabolise the carcinogen to a form which damages DNA.Thus replication occurs in the nodules while DNA damage andrepair occur in the surrounding non-neoplastic liver. With therapid growth of nodules there is overall an increase in neoplastictissue, a relative decrease in nonneoplastic tissue, and thusa relative decrease in DNA damage, repair, and induction ofpolymerase ß. Histo-logical examination showed thatby 35 weeks the conversion to neopflasia was virtually complete.These results support the concept that polymerase functionsin de novo replication of DNA, and is induced during cell replication,while polymerase it functions in repair replication, and increasesin activity during chronic damage to DNA. Whether it is inducedby treatment with carcinogens depends on the duration of treatment,and on other processes (e.g. metabolism of the carcinogen) whichtake place during the development of malignancy.     

Detection of in vivo DNA repair synthesis in mouse liver and lung induced by treatment with benzo(a)pyrene or 4-nitroquinoline 1-oxide

We examined in vivo DNA repair synthesis in liver and lung of A/HeJ mice treated with benzo(a)pyrene (BP) or 4-nitroquinoline 1-oxide. To differentiate between the removal of carcinogen metabolite:DNA adducts due to cell turnover and DNA repair, we measured unscheduled DNA synthesis (UDS) in the nonreplicating DNA fraction. Mice were exposed to bromodeoxyuridine pellets 1 hr prior to carcinogen treatment. Immediately following carcinogen exposure, mice received 4 hourly i.v. doses of [3H]thymidine. Mice were sacrificed 5 hr post-carcinogen treatment, and DNA was isolated. Purified DNA was then separated into newly replicated and nonreplicated DNA by ultracentrifugation in alkaline CsCl gradients. BP induced UDS in the liver at p.o. doses of 0.3 and 3.0 mg/mouse, whereas we failed to detect UDS in the lung. However, 4-nitroquinoline 1-oxide, another lung carcinogen, induced a definite repair response in the lung but not in the liver. It is not clear why mouse lung cells have the capacity to repair 4-nitroquinoline 1-oxide-induced damage to DNA and not the damage induced by BP, since both of these lung carcinogens form bulky adducts with DNA. These results demonstrate that (a) the in vivo disappearance of BP metabolite:DNA adducts from the lung of the A/HeJ mouse is due to cell turnover, whereas the disappearance of adducts from the liver is due, in part, to DNA repair and (b) induction of in vivo UDS after treatment with two different lung carcinogens is both tissue and carcinogen dependent in this mouse strain.     

Persistence of sister chromatid exchanges and in vitro morphological transformation of Syrian hamster fetal cells by chemical and physical carcinogens

The induction of neoplastic cell transformation is closely associatedwith DNA alterations which occur shortly after carcinogen exposure.Sister chromatid exchange (SCE) formation is a sensitive indicatorof carcinogen-DNA interaction and correlates with the inductionof morphological cell transformation. The persistence of lesionsgenerating SCE produced by chemical and physical carcinogensand its relevance to the induction of morphologic transformationwas evaluated in coordinated experiments with cultured Syrianhamster fetal cells (HFC). Exponentially growing HFC were exposedfor 1 h to benzo[a]pyrene (BP), methyl-methanesulfonate (MMS),cis-platinum (II) diaminedichloride (cis Pt II), N-methyl-N'-nitrosourea(MNU), mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), N-acetoxy-2-fluorenyl-acetamide (AcAAF) or u.v. lightirradiated. Cells were incubated for 24 h with 5-bromodeoxyuridine(BrdUrd) required for SCE visualization at 1, 24 and 48 h aftercarcinogen exposure. The induction of morphological transformationwas determined on the quantitative colony assay at 6 days aftercarcinogen treatment. SCE analysis demonstrates that for a periodof 48 h after carcinogen exposure, during which time the cellsundergo at least four replicative cycles, DNA damage generatingSCE induced by all chemical carcinogens either persisted orwas partially removed, whereas u.v.-induced lesions were completelyremoved. An elevated SCE frequency persisted after two additionalcell cycles after treatment with BP, AcAAF or MMC without increasedcell lethality as compared to other carcinogens whose lesionswere completely eliminated during the same period. Althougha correlation between the persistence of SCE and the inductionof transformation was not observed for all carcinogens, thisstudy illustrates that DNA damage generating SCE can persistover several replicative cycles, thus raising the possibilitythat lasting DNA alterations are important for the inductionof neoplastic cell transformation.     

Correlation between incidence of 1,2-dimethylhydrazine-induced colon carcinomas and DNA damage in six genetically different mouse strains

Mice from strains with different susceptibility to the colon-specific carcinogen 1,2-dimethylhydrazine (DMH) were tested for DNA damage in liver, kidney and colon after administration of the compound at a dosage that has been reported to induce a high incidence of adenocarcinoma in the colon of rodents. DNA breaks were evaluated from their elution rate constant according to the alkaline elution technique. We found that 4 h after administration of the carcinogen there was a substantial and comparable DNA damage in liver and kidney of all strains examined. Conversely, colon DNA damage was hardly above control levels in the carcinogen-resistant strains. The highest DNA damage was detected in the most susceptible strain and was slightly lower in the two other susceptible strains. We propose that the extent of DNA breakage in a target organ could be one of the factors determining organ-specific and strain-specific susceptibility to DMH.     

Persistence of DNA single-strand breaks and other tests as indicators of the liver carcinogenicity of 1-nitroso-5,6-dihydrouracil and the noncarcinogenicity of 1-nitroso-5,6-dihydrothymine

The cyclic nitrosourea 1-nitroso-5,6-dihydrothymine [(NDHT) 1-nitrosodihydrothymine] was not significantly carcinogenic when it was administered for 1 year in drinking water (206 mg/liter) to MRC-Wistar rats. In acute toxicity tests, ip injection of saline solutions of 1-nitroso-5,6-dihydrouracil [(NDHU) CAS: 16813-36-8; 1-nitrosohydrouracil], a strong liver carcinogen in rats, produced only mild liver toxicity but marked focal degeneration of myocardial fibers. NDHU injected ip in water solution produced subcapsular liver damage. NDHU, but not NDHT, induced unscheduled DNA synthesis in hepatocyte primary cultures. NDHU, NDHT, and methylnitrosourea [(MNU) CAS: 684-93-5; N-methyl-N-nitrosourea], a liver carcinogen only under special conditions, were tested for their ability, when injected ip into rats, to produce liver DNA damage measured as strand breaks by alkaline sucrose gradient centrifugation. The three nitrosoureas produced similar maximum DNA damage of 2.2-3.2 strand breaks/10(8) daltons. Eighty percent of the damage due to NDHU persisted for 7 days, and the damage at that time was significantly greater than that produced by NDHT and MNU. The varying persistence of liver DNA damage may explain why NDHU, but not NDHT, is a liver carcinogen.     

In vitro binding of N-acetoxy-N-2-acetylaminofluorene to DNA in chromatin.

The binding of N-acetoxy-N-2-acetylaminofluorene to DNA in native and partially dehistonized chicken erythrocyte chromatin was studied. The amounts of carcinogen bound to DNA were measured, after removal of proteins with phenol, by using the absorption ratio A305/A260 or by counting the radioactivity of 14C-labeled carcinogen. Measurements of uncovered zones of DNA in chromatin were made by comparison of results obtained with free DNA and with chromatin at increasing ratios of carcinogen/nucleotide. The proportion of DNA accessible to the carcinogen was found to be 15% in native chicken erythrocyte chromatin and about 22% in native calf thymus chromatin. The amount of accessible DNA increases to 55% in chicken erythrocyte chromatin depleted of histones H1 and H5. Formaldehyde unwinding performed on DNA extracted from chromatin after modification showed an increasing number of defects in the double helix with the amount of DNA-fixed carcinogen. At high ratios of carcinogen/nucleotide, the recoveries of DNA (by phenol method) and of histones (by acidic extraction) decreased with increasing ratios. This suggests a covalent linkage between proteins and DNA.     

Starvation for arginine and glutamine sensitizes human diploid cells to the transforming effects of N-acetoxy-2-acetyl-aminofluorene

We have examined the influence of the method of synchronizationon the transformation of normal human diploid fibroblasts treatedin mid-S phase with N-acetylaminofluorene (N-Ac-AAF), and onthe subsequent kinetics of the removal of carcinogen damage.At early times post-treatment, the transformation frequencyin cultures synchronized by a 24 h starvation for arginine andglutamine was enhanced over that of cultures synchronized byrelease from confluence. In cells synchronized by either method,transformation was enhanced at all times when the cells wereincubated after treatment in medium containing high concentrationsof nonessential amino adds, vitamins and serum. Synchronizedcells treated with 3 µM [³H]N-Ac-AAF in mid-S removedsignificantly less DNA damage than cultures treated and heldunder confluent, noncycling conditions. Cells synchronized byamino add starvation removed less damage than cells synchronizedby release from confluence. S-phase treatment of synchronizedcells resulted in a dose-dependent delay of DNA replication.The early, rapid phase of repair was temporally correlated withthe delay in S-phase progression. We condude from these datathat synchrony achieved by amino acid starvation renders humancells more sensitive to N-Ac-AAF-induced transformation in vitrothan does that achieved by release from density-inhibition ofgrowth. The S-phase treated, cyding cells lost only about 10%of their initial carcinogen damage before reinitiation of treatment-ddayedbulk DNA synthesis. These results support the hypothesis thatthe persistence of carcinogen damage in replicating DNA maybe an important factor in cell transformation.     

Correlation of morphological transformation to sister chromatid exchanges induced by split doses of chemical or physical carcinogens on cultured Syrian hamster cells

The relationship between the induction of DNA damage as reflected by sister chromatid exchange (SCE) formation and morphological transformation in exponentially growing Syrian hamster embryo cells was determined quantitatively after split doses of chemical or physical carcinogens. With split doses of carcinogen separated by 2 to 24 hr, only N-acetoxy-2-fluorenyl-acetamide (0.50 microgram/ml) enhanced both SCE induction and transformation when compared to single exposure. Split doses of N-methyl-N'-nitro-N- nitrosoguanidine (0.20 microgram/ml), mitomycin C (50 ng/ml), or ultraviolet light (3.0 J/sq m) were less effective than single exposures, while split doses of methyl methanesulfonate (40 micrograms/ml) caused transformation frequencies similar to a single treatment and decreased SCE frequencies with time intervals greater than 4 hr. Split or single exposures of X-irradiation (200 R) resulted in similar low frequencies of transformation and SCE. Contrasting with these results, a significant potentiation of SCE occurred after split doses of N-methyl-N'-nitro-N-nitrosoguanidine in cultures arrested in G1 with arginine-glutamine-deficient medium or by contact inhibition compared to a single treatment. This response was attributed to the interaction of carcinogen with DNA containing unrepaired damage and demonstrates the importance of the cell cycle phase of the target cell during carcinogen exposure for the induction of SCE by split doses of N-methyl-N'-nitro-N-nitrosoguanidine. The similarity of responses for transformation and SCE induction with split doses of carcinogens suggests that DNA lesions involved in SCE are essential for the initiation of neoplastic development.     

Differential induction of DNA strand breaks by nitrosamines in the rat liver and esophagus

Hepatic and esophageal nuclei were isolated from Sprague-Dawley rats treated with 5 mg/kg dimethylnitrosamine (DMN), 100 mg/kg diethylnitrosamine (DEN) and 2.5 mg and 10 mg/kg methylbenzylnitrosamine (MBN) and subjected to alkaline elution to determine DNA strand breaks and their subsequent repair. Results obtained showed that hepatic nuclei isolated from rats 4 h after treatment by either DMN or DEN had about 60% of the DNA eluting through the filter. However, at 12 h post treatment, while about 50% of the single-strand breaks in dimethylnitrosamine treated rats were repaired, only about 10-15% of such breaks induced by DEN were repaired in the liver. That DEN is a more effective inducer of hepatic preneoplastic lesions could thus be attributed to this slow repair of the DEN induced lesions. Strand breaks were neither induced by DMN in the esophagus nor by MBN in the liver, the nontarget tissues. More surprising, however, was the finding that MBN induced little or no single-strand breaks in its target tissue, the esophagus. Furthermore, there was no evidence for DNA-protein cross-linking or alkali labile sites in the esophageal DNA. The results indicate that DNA damage induced by the initiating carcinogen in the target tissue may not necessarily involve strand breaks.     

Reduction of DNA repair efficiency during the initial phase of rat liver carcinogenesis

Chemically induced DNA fragmentation and unscheduled DNA synthesis (UDS) were determined in hepatocyte primary cultures (HPC) obtained from the entire liver during the development of hyperplastic lesions induced in rats by the following treatment: 200 mg/kg i.p. of N-nitrosodiethylamine (DEN) on day 0; 2 ml/kg i.g. of CCl4 on day 21; dietary administration of 0.02% 2-acetylaminofluorene (2-AAF) during the 3rd and the 4th week, and of 0.05% phenobarbital (PB) from the 6th week. At 4, 5, 6, and 7 weeks after DEN injection, the level of DNA fragmentation elicited by either the activation-independent carcinogen methyl methanesulfonate (MMS) or the activation-dependent carcinogen N-nitrosodimethylamine (DMN) was significantly lower than that in HPC from age-matched normal rats. In the same HPC, a statistically significant decrease was observed in the capability of repairing DNA damage induced by MMS, while the reduction of the DNA repair efficiency did not reach the level of significance after exposure to DMN.     

Differential effect of gestation stage on benzo(a)pyrene-induced micronucleus formation and/or covalent DNA modifications in mice

Benzo(a)pyrene (BP), an environmental carcinogen, binds ubiquitously to the DNA of maternal and fetal tissues (Lu et al., Cancer Res., 46: 3046-3054, 1986). These studies further investigated the effect of gestation age on the induction of genetic damage by BP. Timed-pregnant ICR mice were treated with one dose of BP on various days of gestation and sacrificed 24-120 h after treatment. At the molecular level, BP covalently bound to the DNA of adult bone marrow and fetal liver of mice at all gestation ages. Compared to the nonpregnant mice (adduct level = 15 adducts/10(8) bases), the adduct levels in the pregnant adult bone marrow were decreased up to 50% during early gestation (days 3-9) and then increased steadily to a 4-fold excess over nonpregnant values during late gestation (days 15-18). In the fetal liver, adduct levels exhibited little variation (3-4 adducts/10(8) bases) between days 11 and 15 of gestation and then increased sharply to 14 adducts/10(8) bases after day 16. At the cellular level, a higher percentage of polychromatic RBCs from adult and fetal mice after BP treatment contained micronuclei (MN) than controls (solvent or untreated). Bone marrow from pregnant mice exhibited greater increases in the formation of MN during early gestation (days 3-9) relative to late gestation (days 15-18), compared to the nonpregnant mice. In the fetuses, the amounts of MN formed were higher than those found in the adult nonpregnant or maternal mice, but these amounts decreased with gestation progression. Thus, MN induction with gestation progression differed from DNA adduction in adults and fetuses. In addition, the dose and time responses of MN formation also differed from those of covalent DNA modifications, when analyzed in the bone marrow of pregnant mice treated on gestation day 5. Collectively, our results showed that pregnancy and development modulate different types of genetic damage in different ways. Fetal tissues may be more sensitive than maternal tissues to genetic damage. Factors in addition to DNA adduct formation may be responsible for MN induction.     

Accumulation and persistence of DNA adducts in respiratory tissue of rats following multiple administrations of the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone

4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major nitrosamine formed in tobacco smoke, induces a high incidence of lung, liver, and nasal cavity tumors in rats. Since alpha-hydroxylation of NNK by target tissues can lead to the generation of a methylating agent, the formation and removal of 7-methylguanine and the promutagenic lesions O6-methylguanine (O6mGua) and O4-methyldeoxythymidine were determined over 12 days of NNK administration to rats (100 mg/kg/day). DNA alkylation was greatest in the nasal mucosa, followed by liver and lung after 1 dose of NNK. No DNA adducts were detected in kidney and brain under these conditions. The concentration of O6mGua increased steadily in lung throughout the treatment regimen, while O6-methylguanine-DNA methyltransferase decreased to less than 5% of control. The concentration of O4-methyldeoxythymidine in lung DNA reached a steady state after 4 days of carcinogen treatment. After NNK treatment was discontinued, O6mGua persisted, while O4-methyldeoxythymidine was removed rapidly in the lung, suggesting that different repair pathways exist for the removal of these adducts in vivo. In hepatocytes, nonparenchymal cells, and nasal mucosa, O6mGua concentrations were maximal after 1-2 days and declined by 50-80% during the remaining 10 days of treatment. The decrease in O6mGua levels in nasal mucosa paralleled a decline in O6-methylguanine-DNA methyltransferase activity and was associated with marked cytotoxicity to Bowman's glands, portions of the lateral nasal gland, and the olfactory and respiratory mucosa during carcinogen treatment. In contrast, the decline in O6mGua in hepatocytes was attributed to the induction of O6-methylguanine-DNA methyltransferase activity, since an 18-fold reduction in the ratio of O6mGua:7-methylguanine was observed over the 12 days of treatment. These studies have demonstrated a marked accumulation of promutagenic DNA adducts in target tissues during repeated exposure to NNK.     

Long-term persistence of nitrosamine-induced structural damage to heterochromatic DNA

Different levels of damage and repair to eu- and heterochromatic DNA from the livers of rats receiving a dose of 10 mg/kg N-nitrosodimethylamine (NDMA) were apparent. Preincorporated 3H-thymidine was lost rapidly from euchromatic DNA but persisted in the heterochromatic fraction. Persistent damage, determined as single-stranded regions binding to benzoylated DEAE-cellulose (BD-cellulose), was evident in heterochromatic DNA for up to three months. By subjecting rats treated with NDMA to partial hepatectomy, generation of single-stranded regions in the newly synthesized heterochromatic DNA could be demonstrated. Such structural defects were evident when hepatectomy was performed two months after administration of the carcinogen. These findings indicate that structural damage to nontranscribed DNA is one of the most persistent molecular lesions following exposure to nitrosamines.     

Inhibition of mammary gland tumors by short-term treatment of estradiol-3-benzoate associated with down-regulation of estrogen receptor ERalpha and ERbeta

Epidemiological evidence indicates that estrogens are one of the risk factors of breast cancer. However, there have been reports that pre-pubertal estrogen exposure is related to reduced breast cancer risk. These discrepancies made us investigate the time-point and duration of estrogen exposure. Our studies focus on the effect of estradiol-3-benzoate (EB) on the mammary gland that was exposed to carcinogens. Ninety-six female Sprague-Dawley rats were randomly divided into 6 groups. Animals at 7 weeks of age were injected with 7,12-dimethylbenz[a]anthracene (DMBA) in groups 1, 2 and 3 or N-methyl-N-nitrosourea (MNU) in groups 4, 5 and 6. One week later, the animals were subjected to sustained treatment with 0 micro g (groups 1 and 4), 30 micro g (groups 2 and 5) or 300 micro g (groups 3 and 6) of EB containing pellets for 4 weeks. All animals were sacrificed at 5 weeks or 21 weeks after carcinogen treatment, for the examination of mammary gland differentiation or mammary gland tumors, respectively. At 21 weeks after carcinogen treatment, the incidence of mammary tumors in group 2 was significantly decreased (P<0.05). EB treatment decreased the multiplicity of DMBA- or MNU-induced mammary gland tumors. At 5 weeks after carcinogen treatment, there were increased branchings of the mammary gland, and there was also a decrease of ERalpha and ERbeta in EB treatment groups. Taken together with these results, we conclude that EB has an inhibitory effect on mammary carcinogenesis, and it suggests that this inhibition may be associated with the differentiation of mammary gland and modulation of ERalpha and ERbeta.     

Effect of visible light on benzo(a)pyrene binding to DNA of cultured human skin epithelial cells

The ubiquity of the photosensitive carcinogen benzo(a)pyrene (BP) and visible light in the environment suggests that their interaction might lead to photoproducts harmful to humans. To test the combined impact of these two agents on human epithelial cells, binding of BP to cellular DNA was assessed following treatment of cultures with BP and low-intensity (4.6 watts/sq m) intermittent (12 hr daily, 3 to 5 days) cool white fluorescent light. Light exposure reduced the formation of covalent BP adducts 20-fold (from 150 to 7 pmol BP per mg DNA) in cells treated with 1 microgram BP per ml and completely inhibited cytotoxicity; even with 10 microgram BP per ml, light exposure markedly inhibited cytotoxicity. However, at low BP dosage (0.1 microgram/ml), covalent adducts (2 pmol/mg DNA) to cellular DNA are produced and their formation is not influenced by light. These adducts persisted for at least 7 days following treatment; this observation suggests that chronic low-level exposure of human epithelium to BP may lead to an accumulation of DNA damage.     

Carcinogen-induced repair and binding in the DNA of chronic lymphocytic leukemic lymphocytes.

Unscheduled DNA synthesis (repair) of carcinogen induced damage of the DNA of lymphocytes from 16 normal and 16 chronic lymphocytic leukemic (CLL) subjects was determined quantitatively for a standard dose of 10 micronM N-acetoxy-2-acetylaminofluorene (NA-AAF). Essentially all the CLL cases (15 of 16) had lower NA-AAF induced repair synthesis values than the normal subjects. Concurrent measurements for the levels of 3H-labeled 7,12-dimethyl-benz(a)anthracene (DMBA) to the DNAs of the normal and CLL lymphocytes after 18 h of culturing in 5 micronM DMBA have shown that 14 of 16 CLL cases had reduced levels of DNA bound carcinogen when compared to the normal individuals. Together these results suggest that CLL lymphocytes have a reduced repair synthesis because there is disproportionately less initial carcinogen-induced damage, and thereby, less substrate stimulation of repair enzymatic activity.