Facilitative mechanisms of lead as a carcinogen

Mutation Research 533 (2003) 121–133

Review

Facilitative mechanisms of lead as a carcinogen Ellen K. Silbergeld∗ Department of Environmental Health Sciences, Johns Hopkins University, Bloomberg School of Public Health, 615 N Wolfe Street, Baltimore, MD 21205, USA Received 5 May 2003; received in revised form 7 July 2003; accepted 7 July 2003

Abstract The carcinogenicity of lead compounds has received renewed attention because of continuing environmental and occupational sources of exposure in many countries. The epidemiological evidence for an association between lead exposures and human cancer risk has been strengthened by recent studies, and new data on mechanisms of action provide biological plausibility for assessing lead as a human carcinogen. Both epidemiological and mechanistic data are consistent with a facilitative role for lead in carcinogenesis, that is, lead by itself may not be both necessary and sufficient for the induction of cancer, but at a cellular and molecular level lead may permit or enhance carcinogenic events involved in DNA damage, DNA repair, and regulation of tumor suppressor and promoter genes. Some of these events may also be relevant to understanding mechanisms of lead-induced reproductive toxicity. © 2003 Elsevier B.V. All rights reserved. Keywords: Lead; Cancer; DNA repair; Zinc finger loop proteins; p53; Oxidative damage

1. Introduction The issue of lead carcinogenicity is of great current interest in science and public health policy. Lead exposures remain significant for many populations world wide, despite the major restrictions on certain uses of lead, such as gasoline additives. These interventions have been based upon the extensive epidemiologic evidence, and supporting mechanistic research, on the noncancer toxic effects of lead, particularly its developmental neurotoxicity [1]. Nevertheless, consideration of lead as a carcinogen is important in light of continuing occupational exposures encountered by adults in a range of industries from microelectronics to building demolition. The allowable blood lead level for workplace exposure in most countries is ∗ Tel.: +1-410-955-8678; fax: +1-443-287-6414. E-mail address: [email protected] (E.K. Silbergeld).

40–50 ␮g/dl, or four to five times that recommended for children [2]. Thus, it is appropriate that the carcinogenicity of lead is under review at present both by the National Toxicology Program in the US and by the International Agency for Research on Cancer. Recent epidemiological data provide increasing evidence that environmental as well as occupational lead exposures may be associated with increased risks of cancer (for reviews [3,4]). For example, our recent analysis of population-based data in the US indicated a significant association between blood lead levels >20 ug/dl and increased risks of premature mortality, primarily due to increased deaths due to cancer and heart disease [5]. In animal models, lead is a reproducible carcinogen [6]. However, policy change based upon this evidence has been impeded until now for several reasons. Of relevance to this review, there has been a lack of mechanistic hypotheses to explain how lead causes cancer at the levels of exposure commonly

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found in occupational and environmental settings. This review focuses on recent mechanistic research suggesting that lead may act in the process of carcinogenesis by increasing the likelihood of fixed damage to DNA, either by inhibiting DNA repair (reviewed by [7]) or by displacing zinc in DNA binding proteins [4]. Both of these events are important in protecting DNA from mutagenic insult. These mechanistic hypotheses would predict that lead is a “facilitative” or “permissive” carcinogen, permitting or augmenting the genotoxic effects of other exposures. Thus, sites for cancer in lead-exposed populations may vary, depending upon its interactions with the direct targets of genotoxic chemical co-exposures and/or on the organs in which lead is preferentially accumulated by the same physiological mechanisms that govern the metabolism of calcium or zinc, such as bone, liver, and prostate [8,9]. In this paper, the epidemiological and experimental studies of lead carcinogenicity are reviewed selectively for evidence in support of this hypothesis, that is, interactions between lead and other exposures. Following this, I discuss the evidence for the current hypotheses for the mechanism of lead as a facilitative carcinogen, and conclude with comments on future research needs.

2. Epidemiological evidence for interactions of lead with other carcinogens Many of the cohort studies of lead and cancer, mostly among lead-exposed workers, have been limited by a failure to identify and control for covariates, especially co-exposures to carcinogens such as cigarette smoke or other metals (notably cadmium and arsenic, which are often found together with lead in smelting and mining facilities) [10]. However, it is possible to consider these co-exposures from a different perspective, as a source of evidence related to possible effect modification of any observed lead/cancer association, suggestive of interactions between lead and these other exposures. For example, interactions were noted between lead and arsenic in increasing risks of lung cancer in smelter workers [11,12], and co-exposures to lead and engine exhaust were also associated with increased risks of lung cancer in workers [13]. Potential interactions

Table 1 All-cause, circulatory disease, and cancer mortality by lead level Mortality

Lead level (␮g/dl) 10–19

20–29

Crude All-cause Circulatory disease Cancer

1.40 (1.16–1.69) 1.27 (0.97–1.57) 1.95 (1.28–2.98)

2.02 (1.62–2.52) 1.74 (1.25–2.40) 2.89 (1.79–4.64)

Sex and age adjusted All-cause Circulatory disease Cancer

1.24 (0.97–1.57) 1.11 (0.87–1.42) 1.70 (1.04–2.78)

1.72 (1.40–2.16) 1.48 (1.10–2.01) 2.39 (1.46–3.90)

Multivariate adjusteda All-cause Circulatory disease Cancer

1.17 (0.90–1.52) 1.10 (0.85–1.43) 1.46 (0.87–2.48)

1.46 (1.14–1.86) 1.39 (1.01–1.91) 1.68 (1.02–2.78)

For purposes of analysis, the reference group is the lead level less than 10 ␮g/dl. a The multivariate model includes the following covariates: age, sex, race, education, income, smoking, body mass index, exercise, and location.

between lead and smoking were directly considered in our recent follow-up study on mortality in the NHANES II cohort (a population-based study of lead exposures and other health risks and indicators in the US conducted from 1976 to 1980). We reported a significant increase in subsequent all-cause mortality that was positively associated with blood lead levels measured once during the original study period [5]. As shown in Table 1, this overall increase in mortality was largely due to cause-specific increases in cancer and cardiovascular diseases. Because information was also available on smoking at the time of blood lead measurement, we were able to examine potential interactions between these two exposures. This analysis indicates a positive interaction between lead and smoking in increasing the risks of lung cancer [5]. These results differ somewhat from a similar study [14] using the same NHANES II database. Using different statistical modeling, this study found a statistically significant and dose-related increase in the relative risks of all cancer only in women. No test for interactions between smoking and lead exposure was conducted by these investigators. The other studies that permit an examination of interactions have reported on biomarkers of genotoxicity in human populations, rather than on actual

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cancer morbidity or mortality. A recent study of metal co-exposures provides evidence for interactions of lead with other exposures. Hengstler et al. [15] studied DNA single strand breaks and repair capacity in lymphocytes from workers exposed to cadmium, lead, and cobalt. Exposure to lead alone did not increase the odds of DNA single strand breaks, but increases in lead exposure from 1.6 to 50 ␮g/m3 in air, in the presence of constant exposures to cobalt and cadmium, resulted in a 5-fold increase in odds of strand breaks Rajah and Ahuja [16] studied in vivo genotoxic effects of smoking and occupational lead exposure in a small group of printing press workers. Their study consisted of four groups: control non-smokers, control smokers, lead-exposed non-smokers, and lead-exposed smokers. Peripheral blood lymphocytes were used to assess mitotic index and DNA damage (sister chromatid exchange, or SCE). Lead-exposed smokers had the lowest mitotic index, as compared to control smokers and lead-exposed non-smokers. The lead-exposed non-smokers also had significantly lowered mitotic index as compared to non-smoking controls. Smokers in the control and lead-exposed groups exhibited a statistically significant increase in the frequency of SCEs when compared to non-smoking controls. While both lead exposure and smoking affected these markers, the combination of lead and smoking did not further increase the frequency of SCEs as compared to either control or lead-exposed non-smokers. In contrast to these studies, no interaction between lead and smoking was found in a study that assessed micronuclei as a marker for genotoxic damage in lead smelter workers, with a consideration of smoking as another risk factor for genotoxicity [17]. There was a highly significant and dose-related increase in the number of cells containing micronuclei in the lead-exposed group as compared to the control group. Although smoking significantly increased the frequency of micronucleus formation in the non-lead group, the frequency of micronucleus formation was not statistically different between lead-exposed smokers and non-smokers. Another small study [18] evaluated SCEs in workers in a lead–zinc powder factory. The frequency of SCE in lead–zinc exposed subjects was significantly higher than in the controls. The SCE frequency was also significantly increased in non-smoking lead-exposed subjects as compared to non-smoking controls. There was a significant in-

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crease in SCE frequency in control smokers as compared to control non-smokers. The results suggest that a positive interaction between lead and smoking may have occurred, but the data are not presented in way that this can be directly tested. Bilban [19] evaluated several markers of cytogenetic damage in Slovenian miners, who were exposed to both lead and radon, and control subjects with potential co-exposures to both smoking and radon. In these miners, it was impossible to separate radon and lead exposure, since both were present in the mine, but both nonsmoking and smoking control groups were recruited, and approximately half (48%) of the miners were reported as nonsmokers. The controls had significantly lower levels of blood lead (a mean of 7 as compared to 34 ␮g/dl) and no measurable exposure to radon. On this basis, the results can be interpreted to suggest an interaction between lead and exposures to radon and cigarettes, in that smoking miners had the highest frequencies of overall chromosomal structural aberrations (including chromatid breaks, chromosomal breaks, dicentric chromosomes, rings, acentrics, but not sister chromatid exchange), followed by smokers in the control group, and the nonsmoking controls having the lowest levels. The miners were not separately analyzed in terms of smokers and nonsmokers, given the likelihood of second hand smoke exposure in the workplace, such a separation may not have been valid.

3. Evidence for lead interactions in experimental studies There is an extensive literature from rodent bioassays and similar long term studies of lead compounds as carcinogens, going back over 60 years (see [20,21] for reviews of this older literature; see [6] for a review of the newer studies). These experimental studies have involved transplacental/translactational, oral, subcutaneous, intratracheal, intramuscular, and intraperitoneal administration of inorganic lead compounds to rats, mice, and hamsters. Taken together these data have been evaluated as sufficient for designating lead as a probable human carcinogen [4,20]. However, two aspects of most experimental studies have tended to raise questions about inferring more certainty for human cancer risk: first, in most studies

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(particularly those before 1990), relatively high doses were used, and second, the sites of increased tumorigenesis in lead-exposed rodents vary widely. Dealing fully with these concerns is beyond the scope of this paper. However, it is important to note that more recent studies have clearly shown that lead can induce tumors in lung and kidney at lower doses as well, and that renal tumors can occur at doses below those inducing nephrotoxicity (discussed further below; see [22,23]. With regard to the multiple and variable sites of lead-induced tumors and other carcinogenic signs, this is not a random phenomenon since the most common sites across studies are kidney, followed by brain, lung, and prostate. Moreover, variability in tumor site is consistent with the hypothesis of co-carcinogenesis that has been advanced for lead and other metals. There have been several experiments where lead was combined with known chemical carcinogens, as shown in Table 2. All but one study show positive interactions, that is, increased tumor yield or genotoxicity when exposures to lead are combined with a genotoxic carcinogen. Of greatest interest, in the context of the epidemiological studies of lead and smoking reviewed above, co-exposure of lead and benzo(a)pyrene (BAP) significantly increased lung adenomas in hamsters [24]. Co-exposures to these agents can be expected in occupational and environmental settings. This study used lead oxide, which is a form of lead found in smelters and in ambient air (when lead is used in gasoline). Benzo(a)pyrene is a potent carcinogen in cigarette smoke and in some urban ambient air samples. A recent study [25] demonstrated co-exposures to lead and BAP in urban populations. The intratracheal route of administration,

used in this study, models inhalation exposure, which would be a common route of human co-exposures to these two substances. The animals were exposed for 10 weeks and followed for 60 additional weeks, so that exposure was not lifelong. Neither lead oxide or BAP alone induced lung tumors, although lead oxide induced hyperplastic and squamous metaplastic alveolar foci. In animals with combined exposures, 11 adenomas and one adenocarcinoma were found in the lungs. The other studies have measured genotoxic endpoints, rather than tumorigenesis. The results are ambiguous. However, two studies have reported no interactions between lead nitrate and hepatic carcinogens. Columbano et al. [26] reported that a single dose of lead nitrate (100 ␮mol/kg) given intravenously resulted in hepatomegaly, but they found no interactions between lead nitrate and diethylnitrosamine (DENA) [27]. Coni et al. [28] also reported that lead nitrate-induced cell proliferation in liver in Wistar rats. However, when rats were given an initiating dose of DENA and then fed a diet containing 0.03% acetylaminofluorene for 2 weeks prior to lead nitrate or partial hepatectomy, no effect of lead nitrate on tumor yield was observed. Using another set of co-exposures, Nehez et al. [29] tested whether lead could enhance the genotoxic effect of the pyrethroid insecticide cypermethrin. Male Wistar rats were treated for 4 weeks in a five times per week schedule with low doses of cypermethrin alone, or in combination with lead acetate. Treatment with lead acetate resulted in a statistically significant increase in the number of bone marrow cells with numerical aberrations but did not alter the number of structural aberrations. The

Table 2 Lead and co-exposure to chemical carcinogens: experimental studies Animal

Pb

Other exposure

Outcome

References

CD-1 rat SD rat

0.5–1% lead acetate in diet 2.6% ppt lead in DW

2-AAF Ethyl urea and sodium nitrate B(a)P

↑ Gliomas No interaction

Haas et al. [1967] Koller et al. [1985]

↑ Lung adenomas (none seen with lead alone) ↑ Renal tumors

Hiasa et al. [1983]

↑ Renal adeno-carcinomas

Tanner and Lipsky [1984]

Syrian hamsters

and

1 mg/animal lead oxid intra-trach

Wistar rats

1 ppt lead acetate in diet

Fischer-344 rats

400 or 10,000 ppm lead acetate in diet

N-ethyl-N-hydroxy ethylnitrosamene N-(4 -fluoro-biphenyl) acetamide

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combination of cypermethrin and lead acetate caused a statistically significant increase in the total number of aberrant cells as compared to lead-treated or vehicle treated cells, consistent with an enhancement of cypermethrin-induced genotoxicity. Two additional genetox studies have utilized plant systems. Ma et al. [30] studied the synergistic effects of mixtures of lead tetraacetate and arsenic trioxide using the Tradescantia micronucleus assay. Lead tetraacetate or arsenic alone increased the frequency of micronuclei formation but lead and arsenic together exhibited antagonistic actions (which could have reflected co precipitation or other effects on metal uptake by the plants). Also using the micronucleus assay in Tradescantia. Gill and Sandhu [31] examined genotoxic effects of arsenic trioxide, dieldrin, and lead tetraacetate alone and in combination. The clastogenicity of lead tetraacetate was not potentiated by combinations of the chemicals added either to the aqueous or soil media.

4. Mechanistic studies on lead Knowledge of the mechanisms by which metals cause cancer remains uncertain, even for those metals, such as chromium, arsenic, and nickel, for which the evidence of human carcinogenicity is considered adequate [22]. Even though lead has been intensely studied for many years, its carcinogenic mechanisms are not well understood (see [32] for review). For lead, mechanistic hypotheses and data must be considered in terms of dose. At high concentrations, lead can bind to DNA and change its conformation [33], break nucleic acids [34,35]. However, the direct genotoxicity of lead occurs only at cytotoxic doses [36,37]. The effective concentrations and doses required to induce genotoxicity in these and other studies are well above the levels associated with other severe toxic effects of lead, including tissue damage and cytotoxicity. In vitro, at subcytotoxic exposures, lead does not induce DNA protein crosslinks [38] or increase the frequency of sister chromatid exchanges in treated V79 cells [39]. However, more recent studies suggest that lead may induce micronuclei at epidemiologically relevant doses in vitro [40]. Since lead can cause tumors in experimental animals at doses well below those associated with such severe tissue damage [23], the high

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dose events are not likely to be relevant mechanisms of carcinogenesis. Several possible mechanisms have been examined to understand better the carcinogenic properties of lead and the conditions required for this effect. These include chronic nephropathy, mitogenesis, alterations in gene transcription, oxidative damage, and various indirect genotoxic mechanisms. 4.1. Renal toxicity and cancer The kidney is a target organ for both acute and chronic lead toxicity [41]. Toxic effects of lead to the kidney in both humans and experimental animals begins with changes in glomerular function, reflected in proteinuria, followed by acute morphologic changes that may slowly progress to a chronic irreversible nephropathy. The initial morphological changes include formation of lead–protein complexes called nuclear inclusion bodies and ultrastructural changes in cellular organelles, especially mitochondria. These histologic features of nephrotoxicity are similar in workers and experimental animals chronically exposed to lead. The evidence in both humans and experimental animals indicates that there is a threshold for lead nephropathy, although there is considerable debate as to what this level is [41]. In rodents, frank proximal tubular injury occurs at blood lead levels (PbB) of 60 ␮g/dl. However, recent observations in humans indicate that long term exposures to considerably lower PbB levels may increase risks of subclinical nephropathy, as indicated by biomarkers of renal dysfunction such as altered serum creatinine levels and small molecular weight proteins in urine [42,43]. Several studies have now associated increased body burdens of lead with increased risks of end stage renal disease [44]. The view that nephrotoxic exposures to lead are necessary for renal cancer is primarily based on the older experimental literature [20,41] in which very high exposures to lead were utilized to induce tumors. These exposures induce a progression of organ damage, from acute reversible nephropathy, to chronic irreversible nephropathy, to renal adenocarcinoma. However, this progression is not necessary for lead-induced cancer, or even nephrotoxicity. Moreover, it should be noted that this argument in no way explains the induction by lead of tumors in sites other than kidney. Finally, we

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can dismiss the theory that lead causes renal tumors through a purportedly male rat-specific event, the induction of alpha-2 urinary globulin, a small molecular weight protein purportedly secreted uniquely by male rats in response to several types of nephrotoxic injury [45,46]. Inorganic lead compounds cause renal tumors in male and female mice [23]. These data thus refute the concepts that organ damage must precede lead-induced cancer, and that absent such damage, no cancer risk exists. 4.2. Mitogenesis Mitogenesis is a less severe form of target organ response to toxicity, often but not always associated with cell death, which reflects hyperproliferative responses to cell injury. This type of response has been proposed as a general mechanism for nongenotoxic carcinogens [47]. Choie and Richter [48] were among the first to examine cell proliferation in response to lead, focusing on the kidney as a target organ for both carcinogenesis and noncancer toxicity. In their experiments, a single intraperitoneal injection of relatively high dose lead acetate (40 mg/kg) induces a 40-fold increase in cell proliferation in the proximal renal tubular epithelium of adult female Sprague–Dawley rats. Within the short time frame of analysis following this treatment, no renal pathology was observed. A similar increase in the mitotic index (45-fold) was observed in the proximal and distal epithelium of the renal cortices of adult female CD1 mice given a single dose of 5 mg/kg lead acetate. As in the rat study, no detectable microscopic lesions were observed over the short term of observation [48,49]. A similar proliferative response was induced in the liver of adult male Wistar rats by administration of a single dose of 100 ␮mol/kg bw lead acetate. This exposure caused a significant enlargement of the liver, with a 30-fold increase in DNA synthesis measured by labeled thymidine incorporation [26]. However, as noted above, this lead-induced cell proliferation did not interact with genotoxic chemicals or with treatments such as partial hepatectomy to increase tumorigenesis or further signs of carcinogenesis [27,28,50,51]. Thus, it does not appear that proliferation alone induced by lead is sufficient for cancer, nor is it clear that this mechanism is involved in interactions among lead and genotoxic carcinogens.

Other biochemical responses in kidney have been examined. Glutathione-S-transferase P (GST-P), a marker for hepatocarcinogenesis in the rat, is increased in rat kidney and liver following exposure to lead [52,53]. Lead exposure also resulted in increased levels of most GSTs in the kidney, and in GST-P in the liver [54]. These biochemical events may not reflect carcinogenesis, but rather indicate a response to oxidative stress, as discussed below. While GST increases in the kidney were independent of GSH depletion or malondialdehyde (MDA) production (a marker for lipid peroxidation), the observed increases of GST-P1 in liver were correlated with GSH depletion and MDA production [54]. Oberley et al. [55] demonstrated marked induction of specific GST isoforms in specific kidney cell types in female Sprague–Dawley rats and their pups following chronic administration of lead acetate in drinking water. Increases in GSTs preceded irreversible renal damage; however, the authors concluded that the observed renal damage was not related to neoplastic events. 4.3. Oxidative damage to DNA A general mechanism for metal toxicity involves the generation of free radicals, either through the depletion of endogenous cellular antioxidants such as glutathione, or the production of radical oxygen species [56]. As noted above, lead exposure can result in glutathione depletion and upregulation of GST enzymes, often accompanied by markers of oxidative stress such as malondialdehyde [54]. Lead has been shown to increase hydrogen peroxide production in cells [57,58]. Another mechanism of free radical generation and adduct formation may involve aminolevulinic acid (ALA), the heme precursor whose levels are elevated by lead exposure through feedback disinhibition of the enzyme ALA synthase [59]. ALA can generate free radicals [60] and has been shown to cause oxidative damage to DNA in Chinese hamster ovary cells in vitro through the formation of 8-OHdG adducts [61]. The role of oxidative damage to DNA in human lead toxicity is also supported by a recent study of 7,8-dihydro-9-oxoguanine adducts in lymphocytes collected from persons exposed environmentally to metals, including lead, chromium, cadmium, and nickel [62].

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Lead may also induce oxidative damage by facilitating or augmenting the effects of hydrogen peroxide. Roy and Rossman [37] found that treatment of supercoiled plasmid DNA with lead acetate for 30 min did not induce nicks or strand breaks. However, addition of hydrogen peroxide to the lead containing reaction mixture induced DNA damage, including nicks and strand breaks. The authors suggested that lead damages DNA indirectly by participating in a Fenton reaction to generate hydroxyl radicals in the presence of H2 O2 . Yang et al. [58] also studied effects of lead on supercoiled plasmid DNA strand breaks. To determine whether H2 O2 was involved, DNA was treated with a lower concentration of lead in the presence and absence of H2 O2 . Under these conditions, neither lead acetate or H2 O2 alone induced nicks or strand breaks in the plasmid. In combination, there was an increase in DNA strand breaks. The singlet oxygen scavengers, sodium azide, and 2,2,6,6-tetramethyl-4-piperidone (TEMP), inhibited lead-induced DNA strand breakage more efficiently than the hydroxyl radical scavengers, mannitol and 5,5-dimethyl-1-pyrroline 1-oxide. Deuterium oxide, a singlet oxygen enhancer, increased lead-induced DNA strand breakage, while catalase and superoxide dismutase did not protect DNA from lead-induced breaks. The interaction of lead and H2 O2 was inhibited by sodium azide, EDTA, catalase and glutathione (suggesting independent roles for both oxygen radicals and metal), and potentiated by superoxide dismutase and mannitol. To further examine the role of reactive oxygen species/intermediates in DNA damage, studies were performed to determine whether 8-hydroxydeoxyguanosine (8-OHdG) was formed in DNA following exposure to lead in the presence and absence of H2 O2 . Neither lead or H2 O2 alone induced a significant increase in the amount of 8-hydroxyguanine (8-OHdG) adducts in DNA. However, combined treatment of DNA with lead and H2 O2 resulted in a 14.4-fold increase in the amount of 8-OHdG, consistent with the hypothesis that lead induces DNA damage through a Fenton-like reaction and that a singlet oxygen is the principal oxygen species involved with inducing the damage [37]. 4.4. Genotoxic effects and chromosomal damage Lead exposure in humans and animals causes chromosomal, as distinct from genotoxic, damage. There

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are reports of chromosomal aberrations, micronuclei, and increased sister chromatid exchange in studies of lead-exposed workers (reviewed by [32]). However, these effects are not consistently found and are generally reported in occupational cohorts with blood lead levels in excess of current OSHA standards (40 mcg/dl) [63,64]. More recently, Valverde et al. [65] exposed CD-1 adult mice to lead by inhalation (6.8 ␮g/m3 ) for 60 min two times per week for up to 4 weeks. DNA damage was assessed by single cell gel electrophoresis, using cells from nasal septum, femur, testicle, brain, lung, and kidney. After only one exposure, changes in DNA migration in the gel were observed in cells from lung, kidney, liver, and testicle. Quantitative evaluation of DNA migration was observed in all tissues except testicle, with brain and femur showing the greatest change. Yuan and Tang [66] reported DNA damage in the Comet assay in the second and third generation of mice exposed to lead in drinking water. Their et al. [40] recently reported that very low concentrations of lead salts, in the range of blood lead levels found in workers (0.01–1.1 ␮M) can induce micronuclei in V79 Chinese hamster fibroblasts, through inhibition of tubulin assembly, thus confirming earlier observations at much lower concentrations [39].

5. Facilitative mechanisms of lead carcinogenicity The evidence discussed above provides little in the way of defining direct genotoxic mechanisms for lead carcinogenicity. Target organ toxicity does not explain the diverse sites of lead-induced tumors, nor the ability of lead to cause kidney tumors at relatively low exposures. Lead at high doses can induce proliferation, but there is no evidence for an interaction between lead-induced proliferation and co-exposure to mutagens. There is some evidence that oxygen radicals, produced by lead (especially in the presence of hydrogen peroxide) may induce DNA adducts, but no evidence as yet that this event will trigger carcinogenicity. There is also evidence that lead can cause chromosomal damage in animals and humans. Thus, it is appropriate to consider the mechanistic evidence for facilitative mechanisms by which lead may interact with other carcinogenic exposures. The strongest evidence that lead may be facilitative, rather

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than initiating in its carcinogenic effects, comes from studies of co-exposures to lead and direct genotoxic agents. Lead in vitro can increase genotoxicity in cells exposed to ultraviolet (UV) radiation [67], hydrogen peroxide [58], X-rays [68], or some but not all chemical mutagens [26,27,37]. Suggestions of similar interactions between lead and sensitivity to genotoxic agents were reported by Restrepo et al. [64], who exposed lymphocytes from battery workers to X-rays in vitro. These effects may be due to lead inhibition of DNA repair mechanisms or increases in error-prone repair mechanisms, involving such targets as DNA polymerase and RNA synthesis [7]. As reported by Zelikoff et al. [39], lead affects DNA synthesis fidelity in Chinese hamster V79 cells. Hartwig et al. [38] reported that lead in vitro interferes with repair of UV-induced DNA damage. This latter mechanism appears to be a common action of several toxic heavy metals that act to prevent closure of UV-induced single strand DNA breaks [68,69]. Hartwig et al. [38] used Chinese hamster ovary (V79) cells to examine the effect of lead acetate on DNA repair processes related to genotoxicity. Lead acetate did not enhance the mutation frequency of the hprt gene in V79 cells when compared to controls. Likewise there was no increase in the frequency of sister chromatid exchange in lead treated cells. Lead did not induce DNA strand breaks in HeLa cells. However, in all the endpoints tested, lead ions interfered with repair of UV-induced DNA damage. The mutation frequency of UV-irradiated cells was increased in cells pretreated with lead acetate prior to irradiation when compared to UV-treated controls. Pretreatment of cells with lead acetate prior to UV irradiation also significantly increased the frequency of SCE. These in vitro models have been utilized to study potential mechanisms of interaction. Frenkel and Middleton [70] reported that while lead acetate did not inhibit DNA or RNA synthesis in HeLa cells, DNA and RNA synthesis was inhibited in intact nuclei, and HeLa DNA polymerase ␣ and RNA polymerase II were also inhibited by lead. Calsou et al. [71] investigated the effect of several metals, including lead, on nucleotide excision repair in vitro in a whole cell extract from HeLa cells. Lead chloride inhibited DNA damage excision activity of the cell extract in a dose dependent manner. Likewise, lead inhibited the recognition of UV DNA damage by the cell extract.

5.1. Zinc finger loop proteins and DNA Lead is known to bind to zinc-binding sites in many proteins, including several DNA-binding proteins, including protamines and histones, as well as transcription regulators such as Sp1 and TFIIA [4,72–76]. All these proteins contain zinc-binding sites, with similar cys-his motifs, which is a well characterized binding site for lead [59]. Thus many, if not all zinc-binding nuclear proteins can probably also bind lead and thus potentially dysregulate gene expression or inhibit other functions, as first suggested by Sunderman and Barber [77]. The critical role of zinc in conferring appropriate conformational structure has been well demonstrated for these zinc finger loop proteins, including transcription regulators [78,79]. We and others have shown in both in vivo and in vitro experiments that lead can bind at a sulfhydryl and nonsylfhydryl site to human protamine (HP2, a histone-like DNA-binding protein unique to sperm). Displacement of zinc by lead causes conformational change in this protein, as demonstrated by spectroscopy, NMR and circular dichroism analysis [75,76,80]. This conformational change results in reduced binding of this protein to DNA consensus sequences. When HP2 is bound to lead, its subsequent binding to consensus sequences in DNA is significantly reduced [75]. Others have reported similar effects of lead on other zinc-containing DNA-binding proteins [72,73,76]. The interaction of lead with certain DNA binding proteins may be another indirect or facilitative mechanism of carcinogenesis. An important function of protamines and histones (analogous somatic cell nuclear proteins), is to protect DNA [81–83]. If lead-containing nuclear proteins bind less well to DNA, genetic material may become more vulnerable to damage from other sources because of the lowered affinity for DNA observed for lead-containing protamines or histones. This may explain the reported positive interactions between lead and chemical mutagens in vitro [37]. There are data suggesting that DNA-metal-protamine complexes are relatively more sensitive to UV-induced mutagenesis [76]. Liang et al. [84] reported that when nickel or copper was bound to HP2 in place of zinc, single- and double-strand DNA breakage was enhanced as well as oxidative DNA base damage to both purine and pyrimidine

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bases. These authors suggested that the DNA damage induced in the presence of HP2 and metals was related to interactions of HP2 with DNA as well as the redox properties of HP2-metal complexes. Alterations in patterns of gene expression may also result from alterations in the interactions between DNA and nuclear transcription factors in which lead is substituted for zinc. Coni et al. [51] provided evidence that lead exposure may cause differences in patterns of mRNA expression for growth factors following regenerative mitogenesis and proliferative mitogenesis. Following partial hepatectomy or carbon tetrachloride administration, increased expression of c-fos, c-jun, and c-myc were noted; however, following lead nitrate administration, only c-jun mRNA was increased. Further studies by Shinozuka et al. [85] demonstrated that lead nitrate induces a rapid increase in tumor necrosis factor-␣ (TNF-␣), which may be involved in triggering proliferative mitogenesis in the liver. Other proteins related to tumorigenesis have been identified in studies of target organ gene expression in response to lead. While these analyses were undertaken to identify novel molecular mechanisms unrelated to lead carcinogenicity, some of the results are relevant to considering potential mechanisms of carcinogenicity. Bouton et al. [86] undertook a broad analysis of lead induced alterations in gene expression in immortalized rat astrocytes exposed to 10 uM lead acetate or 10 uM sodium acetate (as control). Using the Clontech array, mRNA for 48 genes were upregulated and 12 were downregulated. For instance, lead-exposure downregulated expression of the tumor suppressor DCC [deleted in colon cancer] precursor protein. Using the InCyte microarray (in which 282 were also represented on the Clontech array), mRNA for 34 genes were upregulated and 18 were downregulated. Of these, a significant effect of lead was found for the expression of zinc binding transcription factors (not further identified). In a more limited analysis of gene expression in astrocytes, Li and Rossman [87] reported that lead exposure of glioma cells in culture (at concentrations as low as 100 uM) resulted in downregulation of thrombospondin 1 gene expression, which has been considered to be a tumor suppressor protein. These data suggest the importance of further research on the interactions of lead with expression of genes involved in tumorigenesis.

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Lead–protein interactions may also play an important role at the post-translational level. The tumor suppressor protein p53 is a zinc binding protein, and p53 mutations are found in many cancers [88]. It has been shown that when p53 is exposed to cadmium, the result is a structurally altered form of the protein with functional consequences similar to a mutation of the p53 gene [89,90]. No studies have been undertaken of lead–p53 interactions, but this may be another important event in lead carcinogenicity.

6. Implications of carcinogenicity for understanding lead toxicity Given the increasing weight of evidence for lead as a human carcinogen, and the growing body of literature on plausible biological mechanisms for its carcinogenic properties, it is appropriate to consider the implications of carcinogenicity in the overall evaluation of lead toxicity and human health risk. The ability of lead to bind to zinc-binding proteins and to change conformational structure is a well described mechanism for its inhibition of the heme biosynthetic enzyme ALA dehydratase [59]. Through similar interactions at zinc-binding cys-his motifs, lead can also displace zinc and bind to a large number of zinc finger loop proteins, many of which bind to DNA. Lead interactions with protamines may have implications for male-mediated reproductive and developmental toxicity, since DNA-protamine binding affects chromatin decondensation in sperm [75]. The ability of lead to bind to other DNA binding proteins that function as transcription regulators may affect gene expression. As discussed above, lead can induce alterations in gene expression (both up- and down-regulation were reported in glial cells exposed in vitro to lead) [86]. Among these are changes in the expression of genes encoding growth factors such as TNF-␣ [85]. These effects may well have implications for other health effects associated with lead. Altered expression of the gene encoding TNF-␣ is implicated in several birth defects [91]. Thus, the larger implication of new mechanistic research on lead as a carcinogen relates to its potential actions as a reproductive and developmental toxicant. Clearly, DNA-damaging actions in germ cells may be relevant to adverse reproductive outcomes, which have been described in animal models when

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either the female or the male animal is exposed prior to conception [92,93]. Current research in our lab is exploring the potential of lead to alter expression of imprinted genes that are important in early fetal/placental development. Mutagenesis is also a mechanism for teratogenesis [94,95]. As discussed above, there is evidence for lead as a facilitator of mutagenesis. However, the evidence for lead as a teratogen is not clear, although lead is termed a behavioral teratogen [96], it is not clear that this functional definition necessarily implies genotoxicity. Both experimental and epidemiological studies have reported associations between lead exposure and increased incidence of structural defects in offspring [97–99]. Thus increased understanding of the mechanisms of lead as a carcinogen may provide us with mechanistic hypotheses for further research on the developmental and reproductive effects of lead.

7. Summary Lead presents a challenge to understanding mechanisms of metal-induced carcinogenicity. First, lead toxicity in several target organs, notably the nervous system, is well understood, with substantial congruence of evidence between animal models and humans [1]. Second, the in vivo evidence for lead as a carcinogen in rodent models is conclusive, and a growing body of epidemiological studies confirms the inference of human cancer risk. However, lead is similar to other carcinogenic metals in that its carcinogenic mechanisms remain largely mysterious. Earlier theories of target organ damage, hyperproliferation and hyperplasia, or chromosomal damage, are challenged by recent experimental and epidemiological evidence of carcinogenicity in rodents and humans at lower doses. In the past, it was suggested that injury-induced cell proliferation was the major factor in lead-induced renal cancers [49]; however, more recent studies have clearly demonstrated that lead can increase tumorigenesis at exposures below those inducing cell injury in target organs. There are also inconsistencies between those organs in which lead induces cell proliferation, and those organs in which tumors are induced. For example, lead induces cell proliferation in the liver, but no studies have associated liver tumors with lead exposure, and several studies have shown that lead-induced proliferative mitogenesis does not promote tumorigen-

esis in initiated hepatocytes. Moreover, these hypotheses cannot explain the induction of tumors by lead in other organs, including lung, brain, and prostate. At lower doses, lead compounds still induce tumors in animal models and in human populations as well. Yet at these lower exposures, lead does not appear to be directly genotoxic either in vivo or in vitro. The hypothesis of lead as a facilitator of carcinogenesis is increasingly attractive for these reasons. Lead may facilitate genetic damage through several mechanisms. These include inhibition of DNA synthesis and repair, oxidative damage, and interaction with DNA-binding proteins and tumor suppressor proteins. Interference with DNA synthesis and repair has been suggested as one possible explanation for the interactions of lead with genotoxic and mutagenic co-exposures, including UV radiation, oxygen radicals, and chemical mutagens. These mechanisms may be of particular importance in explaining the interactions between this ubiquitous pollutant, still present at unacceptably high levels in many workplaces and environments, and other chemicals present in the complex mixtures of air pollution and cigarette smoke [25]. The continuing exposure of millions of persons worldwide to lead in the workplace and the general environment compels us to understand the short- and long-term hazards of lead, especially its role in causing or contributing to chronic diseases, including cancer.

References [1] D. Rice, E.K. Silbergeld, Lead neurotoxicity: concordance of human and animal research, in: L.W. Chang (Ed.), Toxicology of Metals, CRC Lewis Publishers, New York, 1996, pp. 659–675. [2] WHO, Environmental Health Criteria: Lead, World Health Organisation, Geneva, Switzerland, 1990. [3] H. Fu, P. Boffetta, Cancer and occupational exposure to inorganic lead compounds: a meta-analysis of published data, Occup. Environ. Med. 52 (2) (1995) 73–81. [4] E.K. Silbergeld, M. Waalkes, J.M. Rice, Lead as a carcinogen: experimental evidence and mechanisms of action, Am. J. Ind. Med. 38 (3) (2000) 316–323. [5] M. Lustberg, E. Silbergeld, Blood lead levels and mortality, Arch. Intern. Med. 162 (21) (2002) 2443–2449. [6] ATSDR, Toxicological Profile for Lead, US DHHS, PHS, Agency for Toxic Substances and Disease Registry, 1999. [7] A. Hartwig, T. Schwerdtle, Interactions by carcinogenic metal compounds with DNA repair processes: toxicological implications, Toxicol. Lett. 127 (13) (2002) 47–54.

E.K. Silbergeld / Mutation Research 533 (2003) 121–133 [8] E.K. Silbergeld, Lead in bone: implications for toxicology during pregnancy and lactation, Environ. Health Perspect. 91 (1991) 63–70. [9] E.A. Platz, K.J. Helzlsouer, Selenium, zinc, and prostate cancer, Epidemiol. Rev. 23 (1) (2001) 93–101. [10] K. Steenland, P. Boffetta, Lead and cancer in humans: where are we now? Am. J. Ind. Med. 38 (3) (2000) 295–299. [11] V. Englyst, et al., Lung cancer risks among lead smelter workers also exposed to arsenic, Sci. Total Environ. 273 (1–3) (2001) 77–82. [12] N.G. Lundstrom, et al., Cumulative lead exposure in relation to mortality and lung cancer morbidity in a cohort of primary smelter workers, Scand. J. Work Environ. Health 23 (1) (1997) 24–30. [13] A. Anttila, et al., Excess lung cancer among workers exposed to lead, Scand. J. Work Environ. Health 21 (6) (1995) 460– 469. [14] A. Jemal, et al., The association of blood lead level and cancer mortality among whites in the United States, Environ. Health Perspect. 110 (4) (2002) 325–329. [15] J.G. Hengstler, et al., Occupational exposure to heavy metals: DNA damage induction and DNA repair inhibition prove co-exposures to cadmium, Carcinogenesis 24 (1) (2003) 63– 73. [16] T. Rajah, Y.R. Ahuja, In vivo genotoxic effects of smoking and occupational lead exposure in printing press workers, Toxicol. Lett. 76 (1) (1995) 71–75. [17] A.K. Vaglenov, et al., Cytogenic monitoring of workers exposed to lead, Cent. Eur. J. Occup. Environ. Med. 1997(3) 298–308. [18] H. Donmez, et al., Increased sister chromatid exchanges in workers exposed to occupational lead and zinc, Biol. Trace Elem. Res. 61 (1) (1998) 105–109. [19] M. Bilban, Influence of the work environment in a Pb–Zn mine on the incidence of cytogenetic damage in miners, Am. J. Ind. Med. 34 (5) (1998) 455–463. [20] IARC, IARC Mongraphs on the Evaluation of Carcinogenic Risks to Humans—Some Metals and Metallic Compounds, World Health Organization, International Agency for Research on Cancer, Lyon, France, 1980, pp. 429–448. [21] IARC, IARC Monographs on the evaluation of carcinogenic risks to humans—lead and lead compounds: lead and inorganic lead compounds (Group 2b) organolead compounds (Group 3b), World Health Organization, International Agency for Research on Cancer, Lyon, France, 1987b, pp. 230–232. [22] L.A. Poirier, et al., Inhibition by magnesium and calcium acetates of lead subacetate- and nickel acetate-induced lung tumors in strain A mice, Cancer Res. 44 (4) (1984) 1520– 1522. [23] M.P. Waalkes, et al., Renal tubular tumors and atypical hyperplasias in B6C3F1 mice exposed to lead acetate during gestation and lactation occur with minimal chronic nephropathy, Cancer Res. 55 (22) (1995) 5265–5271. [24] N. Kobayashi, T. Okamoto, Effects of lead oxide on the induction of lung tumors in Syrian hamsters, J. Natl. Cancer Inst. 52 (5) (1974) 1605–1610. [25] V.M. Weaver, et al., Benzene exposure, assessed by urinary trans, trans-muconic acid in urban children with elevated

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34]

[35]

[36] [37] [38]

[39] [40] [41] [42]

[43]

131

blood lead levels, Environ. Health Perspect. 104 (3) (1996) 318–323. A. Columbano, et al., Liver cell proliferation induced by a single dose of lead nitrate, Am. J. Pathol. 110 (1) (1983) 83–88. A. Columbano, et al., Cell proliferation and promotion of rat liver carcinogenesis: different effect of hepatic regeneration and mitogen induced hyperplasia on the development of enzyme-altered foci, Carcinogenesis 11 (5) (1990) 771–776. P. Coni, et al., Different effects of regenerative and direct mitogenic stimuli on the growth of initiated cells in the resistant hepatocyte model, Jpn. J. Cancer Res. 84 (5) (1993) 501–507. M. Nehez, R. Lorencz, I. Desi, Simultaneous action of cypermethrin and two environmental pollutant metals, cadmium and lead, on bone marrow cell chromosomes of rats in subchronic administration, Ecotoxicol. Environ. Saf. 45 (1) (2000) 55–60. T.H. Ma, et al., Synergistic and antagonistic effects on genotoxicity of chemicals commonly found in hazardous waste sites, Mutat. Res. 270 (1) (1992) 71–77. B.S. Gill, S.S. Sandhu, Application of the Tradescantia micronucleus assay for the genetic evaluation of chemical mixtures in soil and aqueous media, Mutat. Res. 270 (1) (1992) 65–69. F.M. Johnson, The genetic effects of environmental lead, Mutat. Res. 410 (2) (1998) 123–140. I. Smirnov, R.H. Shafer, Lead is unusually effective in sequence-specific folding of DNA, J. Mol. Biol. 296 (1) (2000) 1–5. R.S. Brown, et al., Pb(II)-catalysed cleavage of the sugar-phosphate backbone of yeast tRNAPhe--implications for lead toxicity and self-splicing RNA, Nature 303 (5917) (1983) 543–546. A. Wedrychowski, W.N. Schmidt, L.S. Hnilica, The in vivo cross-linking of proteins and DNA by heavy metals, J. Biol. Chem. 261 (7) (1986) 3370–3376. A. Hartwig, Current aspects in metal genotoxicity, Biometals 8 (1) (1995) 3–11. N.K. Roy, T.G. Rossman, Mutagenesis and comutagenesis by lead compounds, Mutat. Res. 298 (2) (1992) 97–103. A. Hartwig, R. Schlepegrell, D. Beyersmann, Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells, Mutat. Res. 241 (1) (1990) 75–82. J.T. Zelikoff, et al., Genetic toxicology of lead compounds, Carcinogenesis 9 (10) (1988) 1727–1732. R. Thier, et al., Interaction of metal salts with cytoskeletal motor protein systems, Toxicol. Lett. 140/141 (2003) 75–81. R.A. Goyer, Lead toxicity: current concerns, Environ. Health Perspect. 100 (1993) 177–187. J.A. Staessen, et al., Impairment of renal function with increasing blood lead concentrations in the general population. The Cadmibel Study Group, N. Engl. J. Med. 327 (3) (1992) 151–156. H.A. Roels, P. Hoet, D. Lison, Usefulness of biomarkers of exposure to inorganic mercury, lead, or cadmium in controlling occupational and environmental risks of nephrotoxicity, Ren. Fail. 21 (3/4) (1999) 251–262.

132

E.K. Silbergeld / Mutation Research 533 (2003) 121–133

[44] R.P. Wedeen, Bone lead, hypertension, and lead nephropathy, Environ. Health Perspect. 78 (1988) 57–60. [45] W.M. Kluwe, K.M. Abdo, J. Huff, Chronic kidney disease and organic chemical exposures: evaluations of causal relationships in humans and experimental animals, Fundam. Appl. Toxicol. 4 (6) (1984) 889–901. [46] L.D. Lehman-McKeeman, D. Caudill, Biochemical basis for mouse resistance to hyaline droplet nephropathy: lack of relevance of the alpha 2u-globulin protein superfamily in this male rat-specific syndrome, Toxicol. Appl. Pharmacol. 112 (2) (1992) 214–221. [47] S.M. Cohen, L.B. Ellwein, Genetic errors, cell proliferation, and carcinogenesis, Cancer Res. 51 (24) (1991) 6493–6505. [48] D.D. Choie, G.W. Richter, Lead poisoning: rapid formation of intranuclear inclusions, Science 177 (55) (1972) 1194–1195. [49] E.J. Calabrese, L.A. Baldwin, Lead-induced cell proliferation and organ-specific tumorigenicity, Drug Metab. Rev. 24 (3) (1992) 409–416. [50] G.M. Ledda-Columbano, et al., Compensatory regeneration, mitogen-induced liver growth, and multistage chemical carcinogenesis, Environ. Health Perspect. 101 (Suppl. 5) (1993) 163–168. [51] P. Coni, et al., Differences in the steady-state levels of c-fos, c-jun and c-myc messenger RNA during mitogen-induced liver growth and compensatory regeneration, Hepatology 17 (6) (1993) 1109–1116. [52] A. Columbano, et al., Modulation of the activity of hepatic gamma-glutamyl transpeptidase, adenosine triphosphatase, placental glutathione S-transferase and adenylate cyclase by acute admsinistration of lead nitrate, Basic Appl. Histochem. 32 (4) (1988) 501–510. [53] T. Suzuki, et al., Activation of glutathione transferase P gene by lead requires glutathione transferase P enhancer I, J. Biol. Chem. 271 (3) (1996) 1626–1632. [54] D.A. Daggett, et al., Effects of lead on rat kidney and liver: GST expression and oxidative stress, Toxicology 128 (3) (1998) 191–206. [55] T.D. Oberley, et al., Effects of lead administration on developing rat kidney II Functional, morphologic, and immunohistochemical studies, Toxicol. Appl. Pharmacol. 131 (1) (1995) 94–107. [56] C.B. Klein, K. Frenkel, M. Costa, The role of oxidative processes in metal carcinogenesis, Chem. Res. Toxicol. 4 (6) (1991) 592–604. [57] M. Ariza, G.N. Bijur, M.V. Williams, Lead and mercury mutagenesis: role of H2 O2 , superoxide dismutase, and xanthine oxidase, Environ. Mol. Mutagen. 1998(31) 352–361. [58] J.L. Yang, et al., Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8-hydroxydeoxyguanosine adduct by lead acetate, Environ. Mol. Mutagen. 33 (3) (1999) 194–201. [59] I.A. Bergdahl, et al., Lead binding to delta-aminolevulinic acid dehydratase (ALAD) in human erythrocytes, Pharmacol. Toxicol. 81 (4) (1997) 153–158. [60] M. Hermes-Lima, B. Pereira, E.J. Bechara, Are free radicals involved in lead poisoning? Xenobiotica 21 (8) (1991) 1085– 1090.

[61] M. Yusof, D. Yildiz, N. Ercal, N-Acetyl-l-cysteine protects against delta-aminolevulinic acid-induced 8-hydroxydeoxyguanosine formation, Toxicol. Lett. 106 (1) (1999) 41–47. [62] H. Merzenich, et al., Biomonitoring on carcinogenic metals and oxidative DNA damage in a cross-sectional study, Cancer Epidemiol. Biomarkers Prev. 10 (5) (2001) 515–522. [63] A. Vaglenov, et al., Occupational exposure to lead and induction of genetic damage, Environ. Health Perspect. 109 (3) (2001) 295–298. [64] H.G. Restrepo, D. Sicard, M.M. Torres, DNA damage and repair in cells of lead exposed people, Am. J. Ind. Med. 38 (3) (2000) 330–334. [65] M. Valverde, et al., Genotoxicity induced in CD-1 mice by inhaled lead: differential organ response, Mutagenesis 17 (1) (2002) 55–61. [66] X. Yuan, C. Tang, The accumulation effect of lead on DNA damage in mice blood cells of three generations and the protection of selenium, J. Environ. Sci. Health Part A Tox. Hazard Subst. Environ. Eng. 36 (4) (2001) 501–508. [67] A. Hartwig, Role of DNA repair inhibition in lead- and cadmium-induced genotoxicity: a review, Environ. Health Perspect. 102 (Suppl. 3) (1994) 45–50. [68] A.B. Fischer, Y. Skreb, In vitro toxicology of heavy metals using mammalian cells: an overview of collaborative research data, Arh. Hig. Rada. Toksikol. 52 (3) (2001) 333–354. [69] R.D. Snyder, G.F. Davis, P.J. Lachmann, Inhibition by metals of X-ray and ultraviolet-induced DNA repair in human cells, Biol. Trace Elem. Res. 21 (1989) 389–398. [70] G.D. Frenkel, C. Middleton, Effects of lead acetate on DNA and RNA synthesis by intact HeLa cells, Biochem. Pharmacol. 36 (2) (1987) 265–268. [71] P. Calsou, et al., Negative interference of metal (II) ions with nucleotide excision repair in human cell-free extracts, Carcinogenesis 17 (12) (1996) 2779–2782. [72] J.S. Hanas, et al., Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins, Mol. Pharmacol. 56 (5) (1999) 982–988. [73] D.H. Petering, et al., Cadmium and lead interactions with transcription factor IIIA from Xenopus laevis: a model for zinc finger protein reactions with toxic metal ions and metallothionein, Mar. Environ. Res. 50 (1–5) (2000) 89–92. [74] N.H. Zawia, Disruption of the zinc finger domain: a common target that underlies many of the effects of lead, Neurotoxicology 21 (6) (2000) 1069–1080. [75] B. Quintanilla-Vega, et al., Lead interaction with human protamine (HP2) as a mechanism of male reproductive toxicity, Chem. Res. Toxicol. 13 (7) (2000) 594–600. [76] W. Bal, K.S. Kasprzak, Induction of oxidative DNA damage by carcinogenic metals, Toxicol. Lett. 127 (1-3) (2002) 55– 62. [77] F.W. Sunderman Jr., A.M. Barber, Finger-loops, oncogenes, Ann. Clin. Lab. Sci. 18 (4) (1988) 267–288. [78] T.V. O’Halloran, Transition metals in control of gene expression, Science 261 (5122) (1993) 715–725. [79] M.A. Searles, D. Lu, A. Klug, The role of the central zinc fingers of transcription factor IIIA in binding to 5 S RNA, J. Mol. Biol. 301 (1) (2000) 47–60.

E.K. Silbergeld / Mutation Research 533 (2003) 121–133 [80] M. Razmiafshari, et al., NMR identification of heavy metalbinding sites in a synthetic zinc finger peptide: toxicological implications for the interactions of xenobiotic metals with zinc finger proteins, Toxicol. Appl. Pharmacol. 172 (1) (2001) 1–10. [81] A.E. Mirsky, B. Silverman, Blocking by histones of accessibility to DNA in chromatin, Proc. Natl. Acad. Sci. U.S.A. 69 (8) (1972) 2115–2119. [82] D. Angelov, et al., Preferential interaction of the core histone tail domains with linker DNA, Proc. Natl. Acad. Sci. U.S.A. 98 (12) (2001) 6599–6604. [83] B. Rydberg, Radiation-induced DNA damage and chromatin structure, Acta Oncol. 40 (6) (2001) 682–685. [84] R. Liang, et al., Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: enhancement of binding and mediation of oxidative DNA strand scission and base damage, Carcinogenesis 20 (5) (1999) 893–898. [85] H. Shinozuka, et al., Roles of growth factors and of tumor necrosis factor-alpha on liver cell proliferation induced in rats by lead nitrate, Lab. Invest. 71 (1) (1994) 35–41. [86] C.M. Bouton, et al., Microarray analysis of differential gene expression in lead-exposed astrocytes, Toxicol. Appl. Pharmacol. 176 (1) (2001) 34–53. [87] P. Li, T.G. Rossman, Genes upregulated in lead-resistant glioma cells reveal possible targets for lead-induced developmental neurotoxicity, Toxicol. Sci. 64 (1) (2001) 90– 99. [88] P.E. Jackson, et al., Specific p53 mutations detected in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry, Cancer Res. 61 (1) (2001) 33–35.

133

[89] P. Hainaut, J. Milner, A structural role for metal ions in the “wild-type” conformation of the tumor suppressor protein p53, Cancer Res. 53 (8) (1993) 1739–1742. [90] P. Hainaut, K. Mann, Zinc binding and redox control of p53 structure and function, Antioxid. Redox. Signal. 3 (4) (2001) 611–623. [91] V. Toder, et al., The role of pro- and anti-apoptotic molecular interactions in embryonic maldevelopment, Am. J. Reprod. Immunol. 48 (4) (2002) 235–344. [92] L. Uzych, Teratogenesis and mutagenesis associated with the exposure of human males to lead: a review, Yale J. Biol. Med. 58 (1) (1985) 9–17. [93] R. Gandley, L. Anderson, E.K. Silbergeld, Lead: male-mediated effects on reproduction and development in the rat, Environ. Res. 80 (4) (1999) 355–363. [94] H. Vainio, Carcinogenesis and teratogenesis may have common mechanisms, Scand. J. Work Environ. Health 15 (1) (1989) 13–17. [95] P.J. O’Brien, et al., Chemical carcinogenesis, mutagenesis, and teratogenesis, Can. J. Physiol. Pharmacol. 74 (5) (1996) 565–571. [96] M. Auroux, Behavioral teratogenesis: an extension to the teratogenesis of functions, Biol. Neonate 71 (3) (1997) 137–147. [97] J.M. Sobotka, R.G. Rahwan, Teratogenesis induced by shortand long-term exposure of Xenopus laevis progeny to lead, J. Toxicol. Environ. Health 44 (4) (1995) 469–484. [98] J.L. Domingo, Metal-induced developmental toxicity in mammals: a review, J. Toxicol. Environ. Health 42 (2) (1994) 123–141. [99] M. Sallmen, et al., Paternal occupational lead exposure and congenital malformations, J. Epidemiol. Community Health 46 (5) (1992) 519–522.