Mutagenesis, Vol. 15, No. 5, 379-383,
September 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Mutation frequency in the lacI gene of liver DNA from lambda/lacI transgenic mice following the interaction of PCBs with iron causing hepatic cancer and porphyria
MRC Toxicology Unit, Hodgkin Building, Leicester University, Lancaster Road, Leicester LE1 9HN, UK
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Abstract |
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The synergistic interaction of iron overload, Ahr genotype and exposure to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1254) in mice leads to hepatic porphyria, oxidative DNA damage and cancer. In humans, hepatocellular cancer is associated with iron overload and hepatic porphyria. Neither the mechanism of hepatic carcinogenesis induced by PCBs in rodents nor hepatocellular cancer induced by iron and porphyria in humans are understood. To test the hypothesis that chronic interaction of iron and PCBs may induce mutagenesis in liver DNA,
/lacI transgenic C57BL/6 mice were given iron dextran (600 mg iron/kg) and then administered Aroclor 1254 in the diet (0.01%) for 7 weeks. Hepatic iron, CYP1A activity and CYP1A1/1A2 protein were elevated >20-fold as a result of iron or Aroclor treatments, respectively, but porphyria with associated histological changes only developed in the combined iron/Aroclor treatment group. /lacI shuttle vectors were isolated from liver genomic DNA and the mutational frequency (MF) in the lacI gene determined. Both iron and Aroclor treatments alone caused significant small increases in MF (1.5- and 1.4-fold, respectively), however, the MF following the combined iron and Aroclor treatment (1.6-fold) was not greater than the additive effects. In contrast, the MF was significantly elevated (4.7-fold) in liver DNA of mice 2 weeks following five daily doses of N-nitrosodimethylamine (4 mg/kg). These studies demonstrate that neither PCBs nor iron overload caused marked point mutations even in a combination regime that leads to oxidative damage and cancer. There was also no strong evidence either that porphyrins or chronic CYP1A1 expression induced by the PCBs after this period caused marked point mutagens or simple deletions. Hence, to understand the PCBs–iron synergism more complex scenarios than point mutations or simple deletions must be invoked. |
Introduction |
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Polychlorinated biphenyls (PCBs) are persistent environmental agents that cause hepatocellular carcinoma in rats and mice (Silberhorn et al., 1990
; Iverson and Grant, 1991). The mechanism of their carcinogenicity is not understood and the compounds give negative results in short-term genotoxic tests (Giri, 1986; Silberhorn et al., 1990; Iverson and Grant, 1991). The chemicals are not thought to form DNA adducts (Whysner et al., 1998) and have been classified as non-genotoxic carcinogens. It seems probable that the initial event in the toxicity of PCBs is mediated through their binding to the aryl hydrocarbon receptor (AHR) (Iverson and Grant, 1991; Enan and Matsumura, 1995). In addition, preloading of Ah-responsive C57BL/10ScSn mice with iron markedly potentiates liver carcinogenesis induced by the PCBs mixture Aroclor 1254, as well as hepatic porphyria, a malfunction of heme biosynthesis (Smith et al., 1990). Ah-non-responsive DBA/2 mice were not susceptible. This suggests that these phenomena may be related mechanistically (Figure 1) and that hepatic carcinogenesis is not just promotion of spontaneous mutations (Smith et al., 1995). Instead of DNA adducts of PCBs, it is possible that the carcinogenic sequence is initiated by an oxidative process which is genetically variable (Faux et al., 1992; Madra et al., 1996). This might be modulated by CYP1A1 expression (Park et al., 1996), which is highly induced by AHR ligands such as non-ortho planar PCBs (Madra et al., 1996), and catalysed by iron. Alternatively, the hepatic porphyrins that accumulate might act as endogenously produced mutagens.
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Transgenic rodents carrying chromosomally integrated bacteriophage
shuttle vectors are a powerful tool for studying mutagenesis in vivo (Dycaico et al., 1994; Morrison and Ashby, 1994; Gorelick, 1995) and a range of mouse hepatocarcinogens have been shown to induce mutations in the lacI gene of the shuttle vector in the liver (Mirsalis et al., 1993; Ross and Leavitt, 1998). Recently, tamoxifen (Davies et al., 1997) and cyproterone acetate (Krebs et al., 1998), which do not induce gene mutations in standard in vitro mutation assays, have been found to induce mutations in vivo in the lacI gene recovered from the livers of /lacI rats. In contrast to PCBs, both compounds form DNA adducts in vivo, which may become fixed as mutations following DNA synthesis. The synergistic interaction of iron with PCBs in mouse liver seemed particularly appropriate as a model to study the role of mutagenesis in neoplasia caused by these chemicals. We show that whilst the liver carcinogen N-nitrosodimethylamine (NNDM) induced a marked increase in mutation of the lacI gene, the individual iron and Aroclor 1254 treatments had only small effects on mutation and a combined treatment that causes liver neoplasia and hepatic porphyria did not cause any further mutations in the lacI gene. |
Materials and methods |
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Chemicals
Iron–dextran complex (50 mg Fe/ml) was obtained from Sigma (Poole, UK). Aroclor 1254 was a gift from Monsanto (USA). Bacto Tryptone was obtained from Difco Laboratories and all other chemicals were obtained from Sigma-Aldrich (Poole, UK). Anti-rat CYP1A1/2 antibody was purchased from Gentest (USA).
Experimental treatments
Male /lacI (Big BlueTM) C57BL/6 mice (6–8 weeks old) were purchased from Stratagene (La Jolla, CA). The animals were maintained under Home Office guidelines as detailed under the Animals (Scientific Procedures) Act 1986. The mice were kept at 19–22°C with a 12 h light/dark cycle in a quarantine area and were fed Rat & Mouse no. 3 maintenance (expanded special quality control) (RM3) diet (S.D.S., Witham, UK) ad libitum supplemented with the additives shown below.
Study 1 was carried out to show that mutagenesis could be detected in the shuttle vector. Mice were divided into two groups. One group received five daily i.p. injections of 4 mg/kg body wt NNDM dissolved in saline. A second group was similarly treated with saline alone. The animals were left untreated for a further 14 days (to allow for the fixation of mutations) prior to killing and the livers were snap frozen in liquid nitrogen and stored at –80°C until DNA isolation.
In study 2 mice were divided up into four groups of six animals. Animals in groups 1 and 3 received single s.c. injections of 600 mg dextran/kg body wt and animals in groups 2 and 4 received single s.c. injections of 600 mg iron/kg body wt (as the 1:1 iron–dextran complex). Five days later mice from groups 1 and 2 were placed on RM3 diet containing 2% corn oil and mice from groups 3 and 4 were placed on RM3 diet containing 0.01% Aroclor and 2% corn oil. The animals were left on their respective diets for 7 weeks prior to termination. This regime eventually produces hepatic tumours in the majority of C57BL/10ScSn mice after 1 year. The 7 week period was chosen as it was felt that porphyria should have started to develop by this time with preliminary histological changes, without severe alterations in pathology which could distort the results (Smith et al., 1990; Madra et al., 1995).
Biochemical analyses
Hepatic iron contents (expressed per g wet tissue), porphyrin levels (expressed as nmol uroporphyrin/g liver) and microsomal ethoxyresorufin deethylase (as an estimate of CYP1A1/2 activity) were determined as described previously (Madra et al., 1996). CYP1A1 and CYP1A2 protein separation was essentially as described in Sinclair et al. (1990) by electrophoresis in a mixed detergent system followed by western blotting using chemiluminescence detection.
Estimation of mutational frequencies
High molecular weight DNA was prepared from liver using a RecoverEaseTM DNA isolation kit (Stratagene). The bacteriophage transgene was recovered from the DNA and packaged into viable phages by incubating with in vitro packaging extract (Transpack, Stratagene). Mutagenesis at the lacI gene was detected as described elsewhere (Kohler et al., 1991; Davies et al., 1997). Mutagenesis at the cII gene was determined as described in Davies et al. (1999). Statistical analysis of mutant frequency (MF) was carried out by analysis of variance (balanced designs) using Minitab version 10 (Minitab Inc., State College, PA).
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Results |
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As a first step to investigate any increase in MF associated with PCBs treatment, a positive mutational genotoxic hepatocarcinogen, NNDM was administered to male
/lacI (Big BlueTM) C57BL/6 mice as five daily doses of 4 mg/kg and the mice left for a further 14 days. This regime induced a MF in the lacI gene significantly higher (4.7-fold) than that obtained following saline treatment alone (Table I). The absolute MF values were very similar to the MF observed previously in mouse liver following administration of another genotoxin, 2-acetylaminofluorene (Ross and Leavitt, 1998). This initial study was commenced prior to the PCBs study and not in parallel, to ensure that in our laboratory the methodology was working well before commitment to the major experiment.
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To investigate the effects of PCBs exposure and iron overload, male
/lacI (Big BlueTM) C57BL/6 mice were administered the PCBs mixture Aroclor 1254 in the diet for 7 weeks with and without prior iron overload. The mouse liver weights as a percentage of body weight at the end of the feeding study were as follows: control 4.8 ± 0.2%; iron treatment 6.5 ± 0.6%; Aroclor treatment 6.6 ± 0.1%; iron and Aroclor treatment 8.7 ± 0.4%. Thus both the iron and the Aroclor treatments significantly (P < 0.01) increased the liver weight compared with that found in control animals. The combined iron and Aroclor treatment significantly increased the liver weights compared with all other treatments (P < 0.01), as observed with C57BL/10ScSn mice treated in a similar manner (Smith et al., 1990).
Figure 2 shows that both the Aroclor and the combined iron/Aroclor treatment markedly increased the hepatic CYP1A1 activity as measured by ethoxyresorufin deethylase activity and this was confirmed by western blotting for CYP1A1 and CYP1A2 protein. In addition, there was marked porphyria in those mice having the combined treatment but at this time of exposure none in the single treatment groups. Furthermore, histologically these iron/Aroclor mice showed early signs of liver lesions. Iron treatment alone showed foci of ironcontaining macrophages throughout the liver, as seen previously with this treatment of the C57BL/10ScSn strain (Smith et al., 1990). Aroclor produced hypertrophy of hepatocytes in the centrilobular region consistent with the increased liver weight. The combination of iron and the PCBs produced not only an additive effect but an apparent increased incidence of mitosis and apoptosis, although these were not assessed quantitatively. The effects were similar but less intense than seen at this time with the C57BL/10ScSn strain. Thus these transgenic mice with the C57BL/6J background showed the porphyric and histological responses to iron and Aroclor combinations previously observed in the C57BL/10ScSn strain (Madra et al., 1996), which eventually led to marked hepatocellular carcinoma (Smith et al., 1990).
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In contrast to the above changes, only small differences in the MF of the lacI gene in the liver were detected for the control, iron, Aroclor 1254 and combined iron and Aroclor treatments (Table II
). There was no evidence of a synergistic interaction between iron and Aroclor being reflected in markedly higher MF rates in the dual treatment group than in the single groups alone. Examination of the data by ANOVA showed that in animals treated with iron there was a significant slight (1.75x10–5 more mutants) increase in MF compared with that seen in the controls. In animals treated with Aroclor there was also a significant slight (1.37x10–5 more mutants) increase in MF compared with the controls. If the effects were additive the combined iron and Aroclor treatment of the mice which led to porphyria and marked histological effects would have had 6.35x10–5 mutants. However, only 5.29x10–5 mutants were observed in this group. Thus, the group having both iron and Aroclor had fewer mutants than expected on an additive basis, though the interaction was not statistically significant, and certainly not the synergistic increase that might be expected. In addition to these studies of the lacI gene, MFs of the cII gene of the phage shuttle vector were also estimated (Davies et al., 1999), but showed no significant increases with the iron or Aroclor treatments (i.e. control 8.0 ± 2.3x10–5; iron 10.5 ± 1.6x10–5; Aroclor 8.2 ± 2.7x10–5; iron/Aroclor 9.6 ± 2.40x10–5), in contrast to a 3.1-fold elevation of MF with NNDM (24.9 ± 5.2x10–5). Comparison with the MFs of the lacI gene seem to suggest that MF of the cII gene is less sensitive, perhaps due to the high MFs of controls in the latter system.
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Discussion |
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To our knowledge, this is the first time that PCBs have been examined for their mutagenic effects in transgenic animals harbouring
shuttle vectors. The combination of iron and Aroclor treatment of C57BL/10ScSn mice gave rise to hepatocellular adenomas in most animals by 8 months and hepatocellular carcinomas by 12 months (Smith et al., 1990). After 2 months of the combined treatment the mitotic rate in the liver was >20-fold higher than in control animals and the emergence of a diploid hepatocyte population was seen very early, within 2 weeks (Madra et al., 1995; Smith et al., 1990). We therefore reasoned that exposure to the combined iron and Aroclor 1254 treatment of the closely related C57BL/6J strain for 7 weeks should have been sufficient time for fixation of mutations. Perhaps our failure to detect a marked increase in MF suggests that any DNA damaging effects of PCBs cannot be detected by examining changes in a short gene such as lacI, where the overwhelming number of mutational events thus far detected are point mutations or small deletions or insertions (Buetner et al., 1996). It is also possible that the small increases in MF are significant over a long exposure period. Similar low MFs were observed in Z#2+/lacI C57BL/6 transgenic mice, which develop a high incidence of hepatocellular carcinoma (Nichols et al., 1998). Particular target cell populations may also be underestimated and the types of mutations in treatment groups could be different and more critical than in controls. However, even so, there was no evidence that MFs were greater in the combined treatment which leads to accelerated cancer development than in the individual treatment groups.
This experiment has also allowed us to test other potential carcinogenic regimes. Firstly, whether porphyrins are endogenous mutagens (Burcham, 1999). Elevated incidences of hepatocellular carcinoma are recorded for both porphyria cutanea tarda and acute intermittent porphyria (Lim and Mascaro, 1995). Porphyrin–DNA adducts have recently been synthesized (Berlin et al., 1998), but if porphyrins are mutagenic, they seem not to be direct acting mutagens in vivo. Secondly, that iron overload could lead to point mutations. Human haemochromatosis has a very high risk of associated liver cancer (Niederau et al., 1985). Clearly, in this model there was only weak evidence for this in iron loaded mice, although over a long period it could be important, and we have observed no increased hepatic tumour incidence in iron overloaded C57BL/10ScSn mice after 18 months (Smith et al., 1989). In preliminary studies hepatic tumours in transgenic mice, derived from the C57BL/6 strain, with iron overload have been reported (Rotenberg and Voland, 1996). Finally, it has been proposed that induction of CYP1A1 could lead to oxidized DNA damage and be a carcinogenic scenario (Park et al., 1996). In fact, both Aroclor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), potent inducers of CYP1A isoforms, do lead to increased oxidized DNA damage in mice (Faux et al., 1992; Shertzer et al., 1998). However, again the evidence from the present experiment is weak that this leads to marked elevation of simple mutagenesis in vivo.
Recent studies have shown that both oxidative stress and PCBs can induce large deletional events in DNA which might not be easily detected with the lacI gene. Arsenite was shown to be mutagenic in an in vitro assay where both intragenic and multilocus mutations were detectable (Hei et al., 1998). Its mode of action was proposed as being mediated through reactive oxygen species and most of the mutant cells had suffered deletional mutations of >3 600 000 bp. The PCB mixture Aroclor 1221 induced homologous recombination following deletion of the 5 kb duplicated sequence present in the hprt gene of Chinese hamster cells (Helleday et al., 1998) and 13.7 kb of the gene in a human lymphoblastoid cell line (Aubrecht et al., 1995). TCDD and other chlorinated PCB mixtures (Aroclor 1221 and Aroclor 1260) have been shown to induce 70 kb deletions in the mouse pun embryo reversion assay by intrachromosomal recombinations (Schiestl et al., 1997). Clearly, to further investigate the synergistic interaction of iron and Aroclor 1254 in C57BL mice, some of these other mutation scenarios should be explored.
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Acknowledgments |
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We would like to thank Dr J.Styles for comments, Dr M.Festing for statistical advice, Dr P.Carthew for histological expertise and S.Kakar for technical assistance.
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Notes |
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1 To whom correspondence should be addressed. Tel: +44 116 252 5617; Fax: +44 116 252 5616; Email: ags5{at}le.ac.uk
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Received on January 7, 2000; accepted on April 14, 2000.
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