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.     

Persistent structural change in replicating hepatic DNA isolated from diethylnitrosamine-treated rats

Structural analysis, by benzoylated O-(diethylaminoethyl) (DEAE)-cellulose chromatography, was made of DNA from the livers of rats receiving 100 mg/kg diethylnitrosamine and subsequently subjected to partial hepatectomy. Under these conditions, different DNA labelling procedures permit damage to be associated with pre-existing or newly synthesised DNA. Persistent single stranded regions could be detected in DNA isolated more than 3 days after carcinogen treatment only if the animals were subjected to hepatectomy. This damage was attributable to lesions impeding DNA replication. Induction of proliferative activity up to at least 14 days after nitrosamine treatment made manifest DNA damage, the extent of which was not decreased as the interval between carcinogen treatment and surgery was increased.     

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.     

Effects of route of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo(c,g)carbazole, benzo(a)pyrene, and 2-acetylaminofluorene

The environmental pollutant 7H-dibenzo(c,g)carbazole (DBC) has been shown to be a potent carcinogen in various mouse tissues, but displays an unusual degree of hepatocarcinogenicity. We have previously reported that in accord with this activity, mouse liver is the target organ for DBC-DNA binding, with total levels being up to 2700 times greater than in extrahepatic tissues after s.c. administration. To elaborate on this finding, we have directly compared the tissue distribution of DNA damage by three diverse aromatic carcinogens, DBC, benzo(a)pyrene (BP), and 2-acetylaminofluorene (AAF). Following a single topical, p.o., or s.c. administration of 80 mumol/kg of test compound to male BALB/c mice, a 32P-postlabeling assay showed the total number of DBC adducts in liver DNA to be 11-138 times that in kidney, lung, or skin DNA. The degree of hepatic adduction varied as a function of the route of administration, with the highest occurring after topical application and the lowest after s.c. injection. The tissue preference for AAF and BP adducts varied with the route of administration and was much less than for DBC adducts, except that topical application of BP gave DNA adduct levels in skin that were 91-218 times greater than in other tissues. For a given tissue and route of administration, DNA adduction by DBC was 1.7- to 950-fold greater than that by BP and AAF, except in skin where the level of DNA adducts from BP was 3 to 4 times that from DBC. We conclude that (a) DBC exhibits an exceptional and unique preference for liver DNA adduction after different routes of administration; (b) DBC is more potent overall than BP or AAF in causing tissue DNA damage; and (c) for each of the three carcinogens, the route of exposure is a much less important factor than the nature of the carcinogen in determining the tissue distribution of covalent DNA damage.     

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.     

DNA damage in liver,colon, stomach,lung and kidney of BALB/c mice treated with 1,2-dimethylhydrazine

DNA single-strand breaks induced in various organs of BALB/c mice by treatment with a single dose of 1,2-dimethylhydrazine (DMH) were studied by means of the alkaline elution method modified in order to allow the evaluation of DNA damage in vivo with no need of radioactive prelabelling. DNA damage was detected in liver, lung, kidney, stomach and colon mucosa, with the liver showing the greatest amount of damage. Its degree was dependent on the dose and route of administration. A differential effect was evident in colon mucosa from Swiss and C57BL/6 mice which are respectively susceptible and resistant to the induction of bowel tumors by DMH. The higher degree of DNA damage found in liver in comparison with colon mucosa is consistent with the previously reported higher degree of DNA methylation, but does not correlate with the specificity of this carcinogen in inducing tumors of the large intestine in mice given repeated subcutaneous injections.     

In vitro antioxidative potential of lactoferrin and black tea polyphenols and protective effects in vivo on carcinogen activation, DNA damage, proliferation, invasion, and angiogenesis during experimental oral carcinogenesis

The present study was designed to evaluate the in vitro antioxidant potential of bovine lactoferrin (bLF) and black tea polyphenols [Polyphenon-B (P-B)] as well as in vivo inhibitory effects on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas. Antioxidant activity was screened using a panel of assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical anion (OH*), superoxide anion (O2*-), and nitric oxide (NO) radical scavenging assays as well as assay for reducing power. The chemopreventive potential of bLF and P-B was assessed in the HBP model based on the modulatory effects on DMBA-induced oxidative DNA damage as well as the expression of proteins associated with carcinogen activation (CYP1A1, CYP1B1), cell proliferation [cyclin D1, proliferating cell nuclear antigen (PCNA), glutathione S-transferase pi (GST-P)], angiogenesis [vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1)], and invasion and metastasis [matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of MMP-2 (TIMP-2)]. Both bLF and P-B showed high radical scavenging activity and reductive potential. Although administration of bLF and P-B alone suppressed DMBA-induced HBP tumors, combined administration of bLF and P-B was more effective in inhibiting HBP carcinogenesis by inhibiting oxidative DNA damage, carcinogen activation, cell proliferation, invasion, and angiogenesis. Our study suggests that the antioxidative property of bLF and P-B may be responsible for chemoprevention of HBP carcinogenesis by modulating multiple molecular targets.     

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.     

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.     

Age-related modification of 7,12-dimethylbenz[a]anthracene binding to rat mammary gland DNA.

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to mammary gland and liver DNA of female Sprague-Dawley rats either 35, 50, or 120 days of age at the time of carcinogen administration was studied. Following a single oral feeding of tritium-labeled DMBA, the level of binding to liver DNA of rats in all 3 age groups was significantly lower, at all times during a 6-week period, than that of binding to mammary DNA. The amount of DMBA bound to liver DNA was a function of the amount of carcinogen administered and not the age of the animal. In contrast, DMBA binding to mammary DNA was dependent on the age of the animal at the time of carcinogen feeding. Furthermore, in the age group with 100% tumor induction (50 days old), DMBA binding increased directly with the amount of carcinogen fed; this was not the case for the other 2 age groups. These results indicated that a significant correlation existed between the age of the rat, the amount of DMBA bound to DNA, and the incidence of mammary tumors following carcinogen feeding.     

Both physiological and pharmacological levels of melatonin reduce DNA adduct formation induced by the carcinogen safrole

Hepatic DNA adduct formation induced by the chemical carcinogen,safrole, was suppressed by both endogenous pineal melatoninrelease and by the exogenous administration of melatonin torats. DNA damage after administration of 100 mg/kg safrole (i.p.)was measured by the PI enhanced 32P-postlabeling analysis method.The RAL (relative adduct labeling) x 10⁷ of carcinogen modifiedDNA in the liver of untreated controls and in safrole treatedanimals killed during the day, at night, after pinealectomyand pinealectomy plus melatonin injection (0.15 mg/kg x 4 ora total of 0.6 mg/kg) was 0, 12.6 ± 0.75, 10.9 ±0.72, 13.6 ± 1.12 and 5.7 ± 0.53 respectively.For the same groups of animals, circulating melatonin levelsat the termination of the study were 31±3, 29 ±2, 276 ±31, 24 ± 1 and 13 950 ± 1016 pg/mlserum respectively. The higher the melatonin concentration inthe serum the lower was DNA adduct formation in the rat liver.Thus, high nocturnal levels of melatonin were protective againstsafrole-induced DNA damage. These findings indicate that thefunctional pineal gland plays an important role in oncostaticactions of carcinogens such as safrole. At physiological levels,melatonin seemed to prevent especially the formation of whatwas referred to as the Nl DNA adduct. Melatonin's ability tosuppress DNA adduct formation may relate to its inhibitory effecton a mixed function oxidase, cytochrome p-450, and on the recentlyidentified hydroxyl radical scavenging capacity of the indole.The oncostatic action of melatonin is also suggested by itsnuclear accumulation and DNA stabilization characteristics.At pharmacological levels melatonin is extremely potent in preventingDNA modification induced by the chemical carcinogen, safrole.     

Decreased NADH-oxidoreductase activities as an early response in rat liver to the carcinogen 2-acetylaminofluorene

Reduced nicotinamide adenine dinucleotide (NADH):ferricyanide reductase and DT-diaphorase specific activity in total homogenates of rat liver are markedly decreased as a very early biochemical event of hepatocarcinogenesis induced by the carcinogen 2-acetylaminofluorene (AAF). A 50 to 75% decrease in NADH:ferricyanide reductase was observed after 1 day of AAF (0.025% in the diet) feeding and persisted throughout a 7-week continuum of AAF administration. Carcinogen added directly to cell extracts had no effect. Similar results were obtained with single injections of either AAF or diethylnitrosamine. Xanthine dehydrogenase was also reduced in liver following AAF administration to nearly the same extent as NADH:ferricyanide reductase and DT-diaphorase. Total NADH-cytochrome c reductase and mitochondrial activity as estimated from succinic dehydrogenase were not affected by carcinogen administration relative to basal dietary controls. The reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase that functions in drug detoxification was elevated. With livers of animals fed 4-acetamidophenol, a hepatotoxin chemically related to AAF, small decreases were noted in NADH:ferricyanide reductase, but not in xanthine dehydrogenase nor in DT-diaphorase. Initial lowering of these activities in the livers of the carcinogen-treated animals is preceded by or concomitant with a reduction in the levels of extramitochondrial pyridine nucleotides known from other studies to result from DNA damage.     

A high pressure liquid chromatography study on the removal of DNA-bound aflatoxin B1 in rat liver and in vitro

Following intraperitoneal administration of [3H] aflatoxin B1 (AFB1) to young adult male rats, there is rapid uptake of the carcinogen by the liver, the target organ for carcinogenesis, leading to DNA covalent binding. Acid hydrolysis of this DNA shows that after 2h, the major DNA adduct is trans 8,9-dihydro-8-(7-guanyl)-9-hydroxy AFB1 (AFB1-gua). By 24h after AFB1 administration the major DNA adduct is no longer AFB1-gua but a product with the identical retention time on h.p.l.c. to 8,9-dihydro-8-(N5-formyl-2',5',6' triamino-4' oxo-N5-pyrimidyl)-9-hydroxy AFB1 (AFB1-triamino-Py). 48h after carcinogen administration, only a small amount of AFB1-gua remains and the major product is AFB1-triamino-Py. The half-life of removal of AFB1-gua is 22h, while AFB1-triamino-Py is much more persistent. In vitro incubation studies on DNA isolated from rats treated 2h previously with [3H] AFB1 show that at pH 7.4 AFB1-gua is the major product released from the DNA with some release of 8,9-dihydro-8,9-dihydroxy AFB1, (AFB1-diol). If more extensively reacted AFB1-DNA is used than that obtained from in vivo administration, then the rate of AFB1-diol release is enhanced while that of AFB1-gua is reduced. It would appear, therefore, that much of the release of AFB1 from DNA in vivo within the first 24h is probably not through a DNA repair process but through chemical release arising from the positively charged N7-guanine. There is considerable conversion of AFB1-gua to AFB1-triamino-Py on in vitro incubation of DNA as well as AFB1-gua and AFB1-diol release. By 24h approximately 66% of the bound AFB1 is in the form of AFB1-triamino-Py and after 48h the conversion is complete. The complex pattern of AFB1-release from DNA may have important consequences in both the induction of mutations and in tumour initiation.     

Effects of Grape Juice in Superoxide Dismutase and Catalase in Colorectal Cancer Carcinogenesis Induced by Azoxymethane

Background: The intestinal mucosa is commonly exposed to oxidant nutrients and carcinogens, which can lead tothe generation of free radicals. The antioxidants present in the diet assume great importance as possible protectiveagents, reducing the oxidative damage. In this way, we evaluated the antioxidant action of grape juice on preneoplasticlesions induced by azoxymethane (AOM) in Wistar rats. Methods: The colorectal carcinogenesis was induced by twointraperitoneal injections of 15mg/kg of AOM in Wistar rats. The animals were divided in 7 groups and treated with1 and 2% concentrations of grape juice before and after carcinogen administration. After euthanasia, the expression ofantioxidant enzymes catalase (CAT), copper-zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxidedismutase (Mn-SOD) CAT, SOD1 and SOD2 were evaluated by immunohistochemistry. Results: AOM decreased theexpression of CAT and Mn-SOD enzymes, but not for Cu/Zn-SOD. We observed an increase expression of CAT andMn-SOD after grape juice administration in some concentrations according to the time of administration of the grapejuice before the carcinogen or just after the carcinogen. Conclusion: Our results suggest an independent action of eachenzyme and a possible antioxidant action of the grape juice components in the diet being able to balance the body toneutralize the superoxide radicals and not leave them in the cell-damaging form.     

Chromosome site-specific immunohistochemical detection of DNA adducts in N-acetoxy-2-acetylaminofluorene--exposed Chinese hamster ovary cells

In these studies a polyclonal antiserum elicited against a carcinogen-DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) in Chinese hamster ovary (CHO) cells exposed to the direct-acting carcinogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) was accomplished by indirect immunofluorescence with an anti-G-C8-AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti-BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N-Ac-AAF for 0.5 h during early S phase, the chromosomal pattern of dG-C8-AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N-Ac-AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.     

Changes in levels of ADP-ribose polymers in rat liver during 2-acetylaminofluorene-induced hepatocarcinogenesis

The exposure of rats to the carcinogen 2-acetylaminofluorene(2-AAF) results in the accumulation of DNA-damaging adducts.The inability of cells to repair such damage adequately is aputative causal event in chemical carcinogenesis. It has beenshown that one cellular response to DNA damage that leads toDNA repair is poly(ADP-ribosyl)ation of nuclear proteins. Toexamine the possible existence of an altered poly(ADP-ribosyl)ationresponse to 2-AAF-mediated damage of rat liver DNA, tissue ADP-ribosepolymer levels were determined during various stages of 2-AAF-mediatedcarcinogenesis. 2-AAF was administered to rats in a discontinuousfeeding regimen comprised of five consecutive cycles, each cycleconsisting of 3 weeks on 2-AAF diet followed by 1 week of recoveryon a control diet without 2-AAF. During cycle one of 2-AAF administration,rat liver ADP-ribose polymer levels increased 3-fold over thatfound in livers of rats fed only the control diet. In contrast,when rats were administered the non-genotoxic liver mitogen4-AAF for one cycle, no significant elevation occurred in ADP-ribosepolymer levels. Elevated ADP-ribose polymer production was alsoobserved during cycles two and three of 2-AAF administration.However, during cycles four and five of 2-AAF administration,a period when rats administered 2-AAF acquire a high risk forhepatocarcinogenesis, an altered pattern of ADP-ribose polymerproduction occurred in rat livers. ADP-ribose polymer levelsin these rat livers remained low, similar to levels found incontrol rat livers, despite the administration of 2-AAF. Whenthe livers from rats fed either one or five cycles of 2-AAFwere analyzed for possible decreases in the levels of tissueNAD+, the substrate for poly(ADP-ribose) polymerase, no changesin relative abundance were found. In addition, analysis of poly(ADP-ribose)polymerase activity showed no decrease at five cycles of 2-AAFadministration. These results indicated that at late stagesof 2-AAF-induced hepatocarcinogenesis, 2-AAF does not inducean expected increase in ADP-ribose polymer levels, and suggestedthat significant changes in DNA repair may occur at a time justpreceding an increased risk for developing liver cancer.     

Liver cell proliferation induced by the mitogen ethylene dibromide, unlike compensatory cell proliferation, does not achieve initiation of rat liver carcinogenesis by diethylnitrosamine

The purpose of this investigation was to determine whether mitogen-induced cell proliferation is as effective as compensatory cell proliferation in achieving initiation of carcinogenesis in rat liver. Male Wistar rats were injected with a single non-necrogenic dose of the hepatocarcinogen diethylnitrosamine (DENA) during the peak of DNA synthesis following the administration of the hepatic mitogen ethylene dibromide (EDB) or a necrogenic dose of CCl4. After subjecting the animals to a promoting procedure, the rats were sacrificed and the initiated hepatocytes were monitored as gamma-glutamyltranspeptidase (gamma-GT) positive foci. The results indicate that while DENA administration during compensatory cell proliferation results in the formation of GT positive foci, no enzyme-altered foci were produced when the carcinogen was given during liver hyperplasia induced by EDB, despite the fact that at the time of carcinogen administration, the extent of cell proliferation, as monitored by thymidine incorporation into DNA, was the same in both the groups.