4-Aminobiphenyl induces liver DNA adducts in both neonatal and adult mice but induces liver mutations only in neonatal mice
Version of Record online: 4 MAY 2005
Copyright © 2005 Wiley-Liss, Inc.
International Journal of Cancer
Volume 117, Issue 2, pages 182–187, 1 November 2005
How to Cite
Chen, T., Mittelstaedt, R. A., Beland, F. A., Heflich, R. H., Moore, M. M. and Parsons, B. L. (2005), 4-Aminobiphenyl induces liver DNA adducts in both neonatal and adult mice but induces liver mutations only in neonatal mice. Int. J. Cancer, 117: 182–187. doi: 10.1002/ijc.21173
- Issue online: 16 AUG 2005
- Version of Record online: 4 MAY 2005
- Manuscript Accepted: 28 FEB 2005
- Manuscript Received: 18 NOV 2004
- genotoxic carcinogen;
- DNA damage;
- Big Blue mice;
The mechanisms underlying the susceptibility of neonatal mice to genotoxic carcinogens were investigated by analyzing the DNA adducts and mutations induced in the livers of neonatal and adult Big Blue transgenic mice by 4-aminobiphenyl (4-ABP), a potent human and rodent carcinogen. Neonatal and adult mice were treated with a regimen of 4-ABP known to induce tumors in neonatal mice. Animals were sacrificed 1 day after the last treatment for DNA adduct analysis and 8 weeks after the last treatment for analysis of lacI and cII mutant frequency (MF). N-(Deoxyguanosin-8-yl)-4-ABP was the major DNA adduct identified in the livers of the 4-ABP-treated mice and levels of this adduct were significantly higher in treated animals than in the controls for both the neonates and adults. Adduct levels for adult females (44.0 ± 4.8 adducts/106 nucleotides) were higher than in neonatal females (25.9 ± 2.2 adducts/106 nucleotides), while adduct levels in adult males (13.5 ± 2.0 adducts/106 nucleotides) were lower than in neonatal males (33.8 ± 4.1 adducts/106 nucleotides). 4-ABP treatment significantly increased the liver cII MFs in both sexes of neonatal mice but not in adult mice. Sequence analysis of cII mutant DNA revealed that 4-ABP induced a unique spectrum of mutations in neonatal mice, characterized by a high frequency of G:CT:A transversion, while the mutation spectrum in 4-ABP-treated adults was similar to that of control mice. Our results indicate that DNA adduct formation by 4-ABP depends as much on sex as it does on age, whereas the conversion of DNA adducts into mutations differed with animal age. These observations suggest that neonates are more sensitive than adults to genotoxic carcinogens because the relatively high levels of cell division in the developing animal facilitate the conversion of DNA damage into mutation. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html © 2005 Wiley-Liss, Inc.
The risk of childhood cancer is surprisingly high, with 1 in 300 males and 1 in 333 females developing cancer by age 20.1 Between 1973 and 1991, the overall cancer incidence in children under age 15 increased 10%.2 Moreover, the incidence of some types of cancer (soft tissue sarcoma, brain cancer and acute lymphocytic leukemia) increased 20–25% during the same period. For a number of cancers, the increase in incidence is highest for young children, especially those in the first 3 years of life.3 While the causes of this increase in cancer incidence are unknown, associations between childhood cancer and environmental exposures have been noted.4, 5, 6 Unfortunately, little is known about how the presence of chemicals in the environment relates to the risk of childhood cancers.7
There is evidence that younger animals are more sensitive to certain carcinogens than older animals in terms of developing chemically induced lymphatic, lung and liver tumors.8 For example, polycyclic aromatic hydrocarbons do not usually induce liver cancer when administered to young adult mice or rats, but do so when given to neonatal animals.9 The mechanism for this increased sensitivity of young animals is not clear, but it has been observed that the neonatal mouse tumorigenicity bioassay is highly sensitive to those carcinogens that exert tumorigenicity via a genotoxic mechanism.10, 11, 12 Therefore, it is reasonable to hypothesize that neonatal animals are more susceptible than adult animals to the induction of mutations induced by genotoxic carcinogens, which results in the higher susceptibility of young animals to carcinogens.
It is generally accepted that the metabolism of chemicals in neonates is less proficient than in adults, and that delayed excretion of a chemical undergoing metabolism results in the prolonged retention of the chemical and some of its metabolites in animal tissue. Such a delay in chemical elimination may result in a greater chance of changing normal cells into mutant and ultimately tumor cells.13 Young animals also have faster cell turnover rates, and the faster rates of cell replication may further increase the sensitivity of neonatal animals to carcinogens. The importance of cell proliferation at the time of carcinogen treatment was demonstrated previously.14 Rapid cell division in the target organs of neonates may cause more rapid conversion of promutagenic DNA adducts into mutations. If metabolism plays a major role in the mutagenic response, we hypothesize that adduct levels would be directly correlated with the mutagenic response irrespective of the age of the animal. If the rate of cell replication is primarily responsible for the higher mutagenic response, we anticipate a higher mutant frequency (MF) in neonates even if the adduct levels were similar in the neonates and adults.
In this study, the Big Blue transgenic mouse model was used to test these alternate hypotheses regarding the sensitivity of neonatal animals to genotoxic carcinogens. The Big Blue model is a powerful approach that can be used to study mutations occurring in any tissue of the mouse. In this system, both DNA adduct formation and mutation induction can be determined simultaneously in vivo. Mutants can be selected, and MFs and types of mutations can be determined for 2 target genes, lacI and cII.15, 16 The aromatic amine, 4-aminobiphenyl (4-ABP), was selected as the test agent for the study for several reasons. 4-ABP is an environmental and occupational contaminant that is a potent carcinogen in both humans17 and the mouse neonatal assay.12 4-ABP is activated to a carcinogen through metabolism,18, 19, 20, 21 and its DNA adduct formation has been characterized. The mutagenicity of 4-ABP has also been characterized in several in vitro and in vivo assay systems.22, 23
Material and methods
Animals and chemical treatment
We followed the recommendations of our Institutional Animal Care and Use Committee for the handling, maintenance, treatment and sacrifice of the animals. 4-ABP and dimethylsulfoxide (DMSO) were obtained from Aldrich Chemical (Milwaukee, WI). The 4-ABP was purified by HPLC to 99.94% and used for all experiments. DMSO was used as the vehicle at approximately 2 μl/g body weight. For treatment of neonatal mice, 4-ABP was dissolved in DMSO. Because larger volumes were used to treat adult mice, they received 4-ABP dissolved in aqueous DMSO (1:1, v:v). The neonatal mice received 1/3 and 2/3 of the total dose or an equivalent volume of DMSO by i.p. injection on postnatal days 8 and 15, respectively. For adult exposures, the mice received 2 doses of an equivalent amount of 4-ABP (on a mg/kg body weight basis) 1 week apart.
In a preliminary dose-response study for adduct formation, 4 groups of 4–7 neonatal male nontransgenic B6C3F1 mice were treated with total doses of 0, 3, 9, or 31 mg/kg 4-ABP. Subsequent studies that compared DNA adduct formation in neonatal and adult Big Blue mice were performed in conjunction with the mutation assays (Fig. 1). Separate groups of 4 neonatal male and female Big Blue B6C3F1 mice and 3–4 4-month-old male and female Big Blue B6C3F1 mice (Stratagene, La Jolla, CA) received the vehicle or 31 mg/kg 4-ABP. The animals were sacrificed 1 day after the second treatment; their livers were removed, frozen quickly in liquid nitrogen and stored at −80°C for analysis of DNA adducts.
To establish a dose response for 4-ABP mutant induction in neonatal mice, groups of 5–6 neonatal male Big Blue B6C3F1 mice were treated with 0, 9, or 31 mg/kg 4-ABP and sacrificed 8 weeks after the last treatment (Supplemental Tables 1–3; supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html). A preliminary experiment indicated that there was a maximum mutagenic response 8 weeks after the last treatment (results not shown). To compare mutant induction in neonatal and adult mice, 5 female neonatal Big Blue B6C3F1 mice and 5–6 male and female 4-month-old Big Blue mice were treated with 31 mg/kg 4-ABP and sacrificed 8 weeks after the last treatment. The mouse livers were removed, flash-frozen in liquid nitrogen and stored at −80°C for lacI and cII mutation assays.
32P-Postlabeling analysis of DNA adducts
Nuclei were prepared from the mouse livers by the method of Basler et al.,24 and DNA was isolated from the nuclei using minor modifications of the method described previously.2532P-postlabeling analyses employed the n-butanol enrichment procedure of Gupta.26 Samples were applied to 10 × 10 cm polyethyleneimine-cellulose TLC plates manufactured by Macherey-Nagel (Alltech Associates, Deerfield, IL). Adducts were resolved by multidirectional chromatography using the following solvents: D1 and D4, 900 mM sodium phosphate (pH 6.8); D2, 3.6 M lithium formate, 8.5 M urea (pH 3.5); D3, 1.2 M lithium chloride, 500 mM Tris-HCl, 8 M urea (pH 8.0). Adducts were visualized with a Storm 860 Imager (Molecular Dynamics, Sunnyvale, CA) and adduct levels were quantified by comparison to a DNA standard modified with N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP) at a level of 62 adducts/108 nucleotides.27
Isolation of liver DNA, packaging of λ phage and mutation assays
High-molecular-weight genomic DNA was extracted from mouse liver using the RecoverEase DNA Isolation Kit (Stratagene) and stored at 4°C until DNA packaging was performed. The λ shuttle vectors containing the target lacI and cII genes were recovered using Transpack Packaging Extract (Stratagene). The resulting phage were used for lacI or cII mutation assays.
The E. coli lacI gene (1,056 bp) can be used as a marker for mutation induction by scoring the color of plaques in the presence of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). The lacI gene encodes the repressor protein for the lacZ gene whose product, β-galactosidase, converts the X-gal to a blue chromogen. Thus, λ phages with a functionally mutated lacI gene make blue plaques among colorless plaques formed by phages with wild-type lacI genes. The lacI mutation assay was performed following Big Blue protocols28 and the manufacturer's instructions for the Big Blue Transgenic Rodent Mutagenesis Assay System (Stratagene). The packaged phage were preadsorbed to SCS-8 E. coli and plated on 25 × 25 cm2 trays containing NZY agar. The plates were incubated overnight at 37°C and the number of mutant (blue) and nonmutant (clear) plaques was determined. The lacI MF was calculated by dividing the number of mutant plaque forming units (pfus) by the total number of pfus analyzed.
The λ phage cII gene encodes a repressor protein that controls the λ lysogenic/lytic cycle. The cII protein induces the expression of the cI and int genes that are required for phage lysogeny. Mutations in the cII gene can be positively selected on a bacterial strain that is deficient for hfI protease. In the hfl−E. coli, phage with active cII genes cannot enter a lytic cycle and only phage with a mutated inactive cII gene can make plaques. The cII assay was conducted following the manufacturer's protocol for the λ Select cII Mutation Detection System for Big Blue Rodents (Stratagene). The plating of the phage was performed with the E. coli host strain G1250. To determine the titer of packaged phage, G1250 bacteria were mixed with 1:100 dilutions of phage, plated and incubated overnight at 37°C (nonselective conditions). When incubated at 37°C, λ phage with either wild-type or mutated cII genes undergo a lytic cycle, resulting in plaque formation. For mutant selection, the packaged phage were mixed with G1250, plated and incubated at 24°C for 42 hr (conditions for cII mutant selection). Using these conditions, λ phage with a wild-type cII gene undergo lysogenization and become part of the developing bacterial lawn, whereas phage carrying a mutant cII gene undergo a lytic cycle and give rise to plaques. The cII MF was derived from the ratio of the number of mutant plaques (determined at 24°C) to the total number of plaques screened (determined at 37°C).
Sequence analysis of cII mutants
A PCR product encompassing the entire cII target was prepared as the template for DNA sequencing as previously described.29 The cII mutant plaques were selected at random from different neonate and adult animals treated with 31 mg/kg 4-ABP and replated at low density to verify the mutant phenotype. Solitary plaques were selected and transferred to a microcentrifuge tube containing 150 μl of sterile distilled water. The tube was placed in boiling water for 5 min and then centrifuged at 12,000 g for 3 min. The PCR included 15 μl of the resulting supernatant and 2 primers (Stratagene) in a 30 μl reaction with 2 × PCR Master Mix (Promega, Madison, WI). The final concentrations of the reagents were 0.5 U/μl Taq polymerase, 1 × reaction buffer (pH 8.5), 1.5 mM MgCl2, 200 μM each dNTP and 0.3 μM each primer. The PCR reaction included a 3-min denaturation at 95°C, followed by 30 cycles of 30 sec at 94°C, 30 sec at 60°C and 30 sec at 72°C, with a final extension time of 10 min at 72°C. The PCR products were purified using a QIAQuick PCR Purification Kit (Qiagen, Chatsworth, CA).
The cII mutant DNAs were sequenced using an ABI Prism Big Dye Terminator Cycle Sequencing Kit and a 377 DNA Sequencer (Applied Biosystems, Foster City, CA). The primers for cII mutation sequencing were the same as those used for the PCR. The sequence data were analyzed with Sequence Navigator software (Applied Biosystems).
Dose-response relationships were assessed by linear regression analysis. Two-way ANOVA followed by a Bonferroni t-test or a Student-Newman-Keul test was used to evaluate the differences in DNA adduct levels and MFs among groups. The mutational spectra were compared using the computer program30 written for the Monte Carlo analysis developed by Adams and Skopek.31
Analysis of DNA adducts
Male B6C3F1 mice were treated on postnatal days 8 and 15 with total doses of 3, 9, or 31 mg/kg 4-ABP; the latter 2 doses are known to produce tumors in neonatal mice.32 Livers were removed from the mice 24 hr after the last treatment and analyzed for DNA adducts by 32P-postlabeling. The major adduct detected was dG-C8-4-ABP, and there was a significant (p < 0.05) dose response, with adduct levels ranging from ∼3 to 16 adducts/106 nucleotides (Fig. 2). Only background adduct levels (∼0.3 adducts/106 nucleotides) were detected in the control B6C3F1 mice.
In a subsequent experiment, neonatal and adult Big Blue transgenic mice were treated with a total dose of 31 mg/kg 4-ABP. The highest adduct level was found in adult female Big Blue mice (44.0 ± 4.8 adducts/106 nucleotides), a value that was significantly (p < 0.05) greater than the other treated groups (Fig. 3). The adduct levels in male and female neonatal Big Blue mice did not differ from one another (33.8 ± 4.1 adducts/106 nucleotides and 25.9 ± 2.2 adducts/106 nucleotides, respectively; Fig. 3) and were significantly (p < 0.05) greater than the value found in adult male Big Blue mice (13.5 ± 2.0 adducts/106 nucleotides; Fig. 3). Only background adduct levels (∼0.2 adducts/106 nucleotides) were detected in the control neonatal and adult Big Blue mice.
Analysis of liver mutation
A dose-response experiment was performed for mutation induction in the liver cII and lacI genes (Fig. 4; primary data in Supplemental Tables 1 and 2). The MFs were measured in male neonatal Big Blue mice treated with 0, 9, or 31 mg/kg 4-ABP and sacrificed 8 weeks after the last treatment. As shown in Figure 4, 4-ABP treatment increased the MFs in a linear dose-responsive manner (p < 0.001). The dose-response trend of mutant induction was similar for the liver cII and lacI mutational targets.
As part of this experiment, female neonatal and adult male and female Big Blue mice were treated with 31 mg/kg 4-ABP and the MFs in the liver cII gene were assessed 8 weeks after the last treatment (Fig. 5; primary data in Supplemental Table 1). The neonates had significantly higher MFs than the adults. In addition, the 4-ABP-induced MF in female Big Blue neonates was significantly higher than the induced frequency in male neonates (p < 0.01). The MFs in adult male and female mice treated with 31 mg/kg 4-ABP did not differ from their corresponding controls (Fig. 5).
Analysis of liver cII mutants by DNA sequencing
Selected liver cII mutants derived from the mutation detection assays were further analyzed by DNA sequencing. The cII mutant plaques were selected at random from different animals. The primary data are presented in Supplemental Table 3 and summarized in Table I. The mutation most commonly induced in mice treated as neonates was G:CT:A transversion. The cII mutational spectra for treated and control neonates were significantly different (p < 0.001). In contrast to the neonatal mice, the most common mutation from the 4-ABP-treated adult mice was G:CA:T transition. The mutational spectrum from the treated adult mice did not differ significantly from the mutational spectra of either untreated neonatal or untreated adult mice (p = 0.126 and 0.17, respectively).
|Type of mutation||Neonate||Adult|
|G:CC:G||17 (16%)||3 (5%)||1 (3%)||3 (9%)|
|G:CA:T||17 (16%)||28 (50%)||13 (36%)||13 (41%)|
|G:CT:A||42 (41%)||12 (21%)||7 (19%)||2 (6%)|
|@ CpG||20 (19%)||24 (43%)||12 (33%)||13 (41%)|
|A:TT:A||4 (4%)||0 (0%)||3 (8%)||0 (0%)|
|A:TC:G||3 (3%)||5 (9%)||0 (0%)||3 (9%)|
|A:TG:C||6 (6%)||1 (2%)||6 (17%)||3 (9%)|
|Frameshifts||13 (13%)||7 (13%)||5 (14%)||7 (22%)|
|Other||1 (1%)||0 (0%)||1 (3%)||1 (3%)|
The relative sensitivity of neonatal organs to carcinogens has been a subject of discussion since 1968.8 Nevertheless, the underlying mechanism(s) for this age-dependent sensitivity remain unclear. One possibility is that the neonatal mice have a lower activity of the metabolic enzymes that convert precarcinogens into inactive conjugates. This hypothesis, however, has never been verified.33 Another possibility is that the damage induced by carcinogens is more efficiently converted into genetic damage in neonates than in adults.
In this study, the treatment of neonatal and adult mice with similar doses of 4-ABP, on a per weight basis, resulted in a significant induction of DNA adducts in neonatal and adult mouse livers, with the extent of adduct formation being dependent on both the sex and age of the animals. For example, adult female mice had higher adduct levels than neonatal female mice, whereas neonatal male mice had higher adduct levels than adult male mice (Fig. 3). Although a significant induction of DNA adducts was detected in both adult and neonatal mice, liver mutations were induced only in the neonates (Fig. 5). Fletcher et al.34 have evaluated the mutagenicity of 4-ABP in the liver of 7-month-old Muta mice and found that 75 mg/kg 4-ABP did not significantly increase the MF in the lacZ gene, whereas 200 mg/kg 4-ABP did. This finding suggests that a considerably higher dose of 4-ABP than used in our experiments would have been required to induce mutations in the livers of adult Big Blue mice. Overall, these data indicate that although there were some differences in the extent of DNA adduct formation in adult and neonatal Big Blue mice, the major difference between the adult and neonatal mice appears to be their relative sensitivity to the mutagenic effects of the adducts, with the neonatal mice being far more sensitive than adults.
These observations suggest that the fixation of DNA damage into mutations is the major factor accounting for the sensitivity of neonatal mice to genotoxic carcinogens such as 4-ABP. DNA replication is a prerequisite for DNA adduct-induced mutagenesis.35 The higher rate of cell proliferation occurring in the neonatal liver, as compared to the adult liver, is likely to potentiate the mutagenic effects of 4-ABP because rapid cell division makes a cell population more vulnerable to the genotoxic effects of chemicals and leaves less time for DNA repair prior to fixation of the damage as a mutation.36, 37 It has been reported that each neonatal liver cell divides an average of 3 times, whereas adult liver cells have less than one division every 3 months.38, 39, 40, 41 Differences in the activities of DNA repair enzymes in neonates and adults may also contribute to age-dependent sensitivity, but currently there is no evidence that DNA damage is repaired more efficiently in adults than in neonates. Consequently, the relatively high rate of cell proliferation in the neonatal liver is likely to be responsible for the increased sensitivity of young mice to the mutagenic and carcinogenic effects of genotoxicants such as 4-ABP.
In carcinogenesis studies with neonatal exposures, similar 4-ABP dose and treatment schedules as used in this study significantly increased liver tumor incidence.12, 42, 43 Identical procedures used in adult animals have not produced comparable responses. Liver tumors were induced in adult mice only when the total 4-ABP dose reached 420 mg/kg.44 These correlations between the induction of mutation and the induction of tumors by 4-ABP in neonatal and adult mice are consistent with mutation being an integral step in carcinogenesis and suggest that mutation in reporter genes can provide early estimates of tumor outcome.
To evaluate the types of mutations induced by 4-ABP and to determine whether the mutation induction by 4-ABP in neonatal mice resulted from clonal expansion due to rapid cell division, mutational spectra from 4-ABP-treated neonatal and adult mice, as well as their controls, were analyzed. The mutational spectrum derived from 4-ABP-treated neonates was dominated by G:CT:A transversion, while the most common mutation from 4-ABP-treated adults and the controls was G:CA:T transition, a typical spontaneous mutation45 (Table I). Although an in vivo 4-ABP-induced mutational spectrum has not been determined previously, our in vivo spectrum is consistent with the types of mutations induced in vitro by 4-ABP or its proximate metabolite, N-hydroxy-4-ABP.46, 47, 48, 49, 50 4-ABP undergoes biotransformation to yield reactive metabolites that are capable of binding covalently to DNA and forming promutagenic DNA adducts.51 The major DNA adduct was identified as N-(deoxyguanosin-8-yl)-4-ABP,20 which has been shown to result in G:CT:A mutation.50 Furthermore, in B6C3F1 mice, 85% of 4-ABP-induced tumors had G:CT:A mutation in H-ras codon 61.43 Our sequencing data also suggest that clonal expansion does not play a crucial role for the MF induced by 4-ABP in neonatal mice. If an initially mutated cell was expanded and resulted in a high MF, we would find a high proportion of sibling mutants. In neonatal mice treated with 4-ABP, however, 103 of 106 cII mutants were independent (Supplemental Table 3).
The levels of 4-ABP DNA adducts were higher in the liver DNA of the female adult mice than in the males, which is consistent with previous reports.52, 53 No such difference, however, was found between the sexes of neonatal mice (Fig. 3). Arylamine carcinogens are N-oxidized followed by conjugation with acetate, sulfate, or glucuronate in the liver. It has been suggested that 4-ABP induces lower DNA adduct levels and tumor incidence in the livers of males relative to females due to the higher chemical conjugation capability of males.54, 55, 56, 57, 58 The similarity of DNA adduct levels induced by 4-ABP in male and female neonatal mice implies a similar level of arylamine metabolism in these mice.
4-ABP increased liver cII MF in both the male and female neonatal mice, with the level being higher in females (Fig. 5). Previous studies indicate, however, that the incidence of 4-ABP-induced liver tumors is higher in neonatal males than neonatal females.42 In general, neonatal treatment of male mice results in higher liver tumor incidences than similar treatments of female mice.33, 59, 60, 61 The tumor-promoting effect of testosterone has been considered the main reason for the greater susceptibility to liver carcinogens in male neonates.62, 63, 64, 65, 66, 67, 68 The initiated cells in male liver may form tumors due to promotion by testosterone, whereas the initiated cells in females do not progress to tumors because of the low amount of testosterone in the female liver.
In summary, treatment of neonatal and adult mice with 4-ABP led to significant increases of DNA adducts. In contrast, neonatal treatment with 4-ABP resulted in a significant increase of liver cII MF, whereas no mutation induction by 4-ABP was found for the adult treatment. The greater susceptibility of neonatal mice to the mutagenicity of 4-ABP may be due to faster cell replication in neonatal than adult liver. If the cell proliferation rates in developing organs facilitate the development of mutations, more rapidly dividing organs in young animals will be more sensitive to the effects of carcinogens. Therefore, proliferation rates in developing organs should be considered when evaluating the carcinogenic potential of childhood exposures and the early life exposures should not simply be prorated over an entire life span when assessing the cancer risks of genotoxic chemicals. Depending on the chemical agent and the target tissue, however, it is probable that cell proliferation will not always be the rate-limiting step in determining age-dependent sensitivity to carcinogens. A recent study on differential susceptibility of age to DNA adduct formation and tumor induction showed an association between the PhIP-DNA adduct levels and the induction of intestinal tumors, with higher PhIP-DNA adduct levels and tumor incidence after exposure on day 12 as compared to day 36.69 Inasmuch as the rates of cell replication in the small intestine do not differ between neonatal and adult animals, the limiting factor for tumor induction in this instance appears to be the extent of adduct formation rather than differences in the rate of cell replication.
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- 17IARC. Summaries and evaluations, supplement 7. Lyon: IARC, 1987.
- 39Cell proliferation and renewal in the mammalian body. In: CameronIL, ThrasherJD, eds. Cellular and molecular renewal in the mammalian body. New York: Academic Press, 1971. 45–85..
- 40Cell numbers vs. age in mammalian tissues and organs. In: CristofaloHV,ed. CRC handbook of cell biology of aging. Boca Raton, FL: CRC Press, 1985. 1–115..
- 51Arylamine-DNA adduct formation in relation to urinary bladder carci-nogenesis and Salmonella typhimurium mutagenesis. In: SugimuraT, KondoS, TakebeH, eds. Environmental mutagens and carcinogens. New York: Alan R. Liss, 1982. 385–96., , , , , .
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