- Email: [email protected]
Effects of carcinogenic metals on gene expression
Toxicology Letters 127 (2002) 63 – 68 www.elsevier.com/locate/toxlet
Effects of carcinogenic metals on gene expression Detmar Beyersmann * Department of Biology and Chemistry, Uni6ersity of Bremen, D-28334 Bremen, Germany
Abstract Six metals and/or their compounds have been recognized as carcinogens: arsenic, beryllium, cadmium, chromium, cobalt and nickel. With the exception of arsenic, the main rote of exposure is inhalation and the main target organ is the lung. Arsenic is exceptional because it also produces tumors of skin and lung after oral uptake. With the exception of hexavalent chromium, carcinogenic metals are weak mutagens, if at all, and their mechanisms of carcinogenicity are still far from clear. A general feature of arsenic, cadmium, cobalt and nickel is their property to enhance the mutagenicity and carcinogenicity of directly acting genotoxic agents. These properties can be interpreted in terms of the ability of these metals to inhibit the repair of damaged DNA. However, because carcinogenic metals cause tumor development in experimental animals even under exclusion of further carcinogens, other mechanisms have to be envisaged, too. Evidence will be discussed that carcinogenic metal compounds alter patterns of gene expression leading to stimulated cell proliferation, either by activation of early genes (proto-oncogenes) or by interference with genes downregulating cell growth. Special reference will be devoted to the effects of cadmium and arsenic on gene expression, which have been studied extensively. Possible implications for occupational safety and health will be discussed. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Carcinogenic metals; Beryllium; Cadmium; Chromium; Arsenic; Nickel; Cobalt
1. Introduction Six metals and or their compounds have been recognized as human carcinogens, i.e. arsenic, beryllium, cadmium, cobalt, nickel and hexavalent chromium by international agencies (International Agency for Research on Cancer, European Union). With the exception of arsenic, the main route of human exposure is inhalation and the main target organ is the lung. In this respect, * Fax: + 49-421-218-7433. E-mail address: [email protected] (D. Beyersmann).
arsenic is exceptional because it produces skin and lung tumors after oral uptake. With the exception of hexavalent chromium [Cr(VI)], carcinogenic metals are weak mutagens, if at all. Cr(VI) is exceptional because it directly reacts with biological material to produce reactive oxygen species which are able to cause DNA damage and gene mutation (De Flora and Wetterhahn, 1989). The mechanisms of action of carcinogenic metals are still far from being elucidated completely. A general feature of arsenic, cadmium, cobalt and nickel is their property to enhance the mutagenicity and carcinogenicity of directly acting
0378-4274/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 4 8 4 - 2
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D. Beyersmann / Toxicology Letters 127 (2002) 63–68
genotoxic agents. Combined with ultraviolet or ionizing irradiation or other DNA damaging agents, these metals are comutagenic and cocarcinogenic (Hartwig, 1995). A generally applicable mechanism of carcinogenicity of arsenic, cadmium, cobalt and nickel seems to be the inhibition of DNA repair enzymes and the consequent enhancement of DNA damage originally caused by other agents or raised spontaneously. However, because compounds of these metals cause tumors in animals under exclusion of further carcinogens, other mechanisms of transformation to malignant growth have to be envisaged, too. Many carcinogenic metal compounds alter patterns of gene expression. Some of these changes lead to deregulated cell proliferation, either by activation of early genes or by interference with genes downregulating cell growth and senescence. Expression of genes may be modified by interference of the metal ions or their reactive metabolites (e.g. reactive oxygen species) with elements of signal transduction cascades such as second messengers, protein kinases and phosphatases or transcription factors. The classical experimental approach investigates the expression of genes of interest by mRNA and or protein analyses whereas a more recent approach assays changes in
Fig. 1. Effects of arsenic on the expression of selected genes.
a multitude of gene activities by DNA array techniques.
2. Arsenic Genetic effects of arsenic are summarized in Fig. 1. Arsenite activated all major mitogen-activated protein kinase pathways in various mammalian cell lines, which is explained by inhibition of the corresponding protein phosphatases (Cavigelli et al., 1996). Arsenite caused enhanced binding of the mitogenic transcription factor AP-1 to DNA, and it activated the expression of the early genes c-fos, c-myc and egr-1, and of the stress genes gadd153 and gadd45 (Simeonova et al., 2000). The activation of transcription factor AP-1 and the induction of some early genes supports the hypothesis that arsenic promotes neoplastic growth through stimulation of cell proliferation. In another study, effects of metals on gene expression regulated by 13 different promoters in a recombinant cell line were investigated (Tully et al., 2000). Arsenate was found to activate the promoters for MTIIA, GSTYa, HSP70, Fos, XRE, NF-kB, GADD153, p53, and CRE. This list includes several genes coding for cytoprotective proteins, namely metallothionein, glutathione-S-transferase, and some stress proteins. Arsenic also interfered with the transcriptional activity of glucocorticoid receptor complexes, thereby decreasing the expression of genes that downregulate cell proliferation in favor of differentiation (Kaltreicher et al., 2001). Other authors observed that arsenite inhibited the activation of the transcription factor NF-kB and the transcription of genes mediated by this factor (El-Sabban et al., 2000; Roussel and Barchowsky, 2000). This effect was found to be caused by an inhibition of I-kB kinase, an enzyme required for the phosphorylation and degradation of the inhibitor I-kB (Kapahi et al., 2000). Because the dominant effect of NF-kB seems to be growth inhibitory (Seitz et al., 1998), the prevention of NF-kB activation by arsenite points to a further link to the stimulation of cell proliferation in arsenic-induced carcinogenesis.
D. Beyersmann / Toxicology Letters 127 (2002) 63–68
Fig. 2. Examples for genes induced by cadmium.
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enod and Jones, 1992; Nicotera et al., 1994; Benters et al., 1996) and to cause a sustained activation of mitogen-activated protein kinases that correlated with the induction of c-fos (Wang and Templeton, 1998). The latter effect was specific for cadmium, because six other metal ions tested were inactive (Ding and Templeton, 2000). The activation of mitogenic signaling pathways offers an attractive hypothesis of cadmium carcinogenesis, which in synergy with the inhibition of DNA repair and in antagonism with the stimulation of cytoprotective mechanisms by cadmium, may explain the complex organ specifity in cadmium carcinogenesis.
3. Cadmium 4. Chromium Cadmium is the toxic metal most extensively studied with respect of gene expression (reviewed by Beyersmann and Hechtenberg, 1997). Genetic effects of this metal are summarized in Fig. 2. Similar to arsenic, cadmium induces at least two types of genes: (1) genes coding for detoxifying and other cytoprotective proteins, i.e. metallothioneins, enzymes of glutathione synthesis, heat shock (stress) proteins, zinc transporter proteins; and (2) early genes resp. proto-oncogenes related to cell proliferation control (c-fos, c-jun, c-myc, egr-1). The concentrations of cadmium required for the induction of metallothionein and protooncogenes are remarkably low in the subtoxic range, i.e. a few mM. At variance, stress (heat shock) proteins are induced by cytotoxic cadmium concentrations, i.e. 10– 30 mM, depending on the cell system. The induction processes of metallothioneins and proto-oncogenes are relatively specific for cadmium. This metal is the strongest inducer of metallothionein promoters. Amongst metal ions, cadmium as well as arsenite is a relatively specific inducer of early genes at low concentrations, whereas the stress response seems to be the consequence of more severe cytotoxic damage by elevated cadmium concentrations. The induction of early genes by cadmium is mediated by interference of its ions with cellular signal transduction mechanisms. Cadmium ions have been shown to interact with cellular calcium homeostasis (The´ v-
Chromate(VI) is the only carcinogenic metal species that directly generates reactive oxygen species by interaction with cellular reductants. Hydroxyl radicals, when generated in proximity to DNA, cause DNA strand breaks and oxidized bases (De Flora and Wetterhahn, 1989). Besides causing direct gene mutations, Cr(VI)-evoked formation of OH radicals has been shown to activate nuclear factor-kB which may stimulate inflammatory processes (Ye et al., 1995). In a commercial DNA array test system employing human hepatoma cells, chromate(VI) at low concentrations of 5–10 mM induced the promoters for c-Fos, HSP70, GADD45, NF-kB, p53, XRE and CRE (Tully et al., 2000). A model for the dual cellular effects of chromate(VI) is depicted in Fig. 3.
5. Cobalt At variance, cobalt(II) does not cause direct oxidative stress, but may undergo Fenton-type redox chemistry with hydrogen peroxide (Fig. 4). The resulting decrease in cellular hydrogen peroxide is believed to affect an oxygen-sensing mechanism regulating the hypoxic reaction of human hepatoma cells (Porwol et al., 1998). This leads to the activation of the hypoxia-inducible factor, a transcription factor which induces the expression
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Fig. 3. Mechanisms of DNA damage and gene induction by chromate(VI).
of various genes including those coding for erythropoiesis but also for several growth factors for angiogenesis (Gleadle et al., 1995). The reactive oxygen species generated from hydrogen peroxide may cause DNA damage and mutation, but the mutagenicity of cobalt is very weak (reviewed by Beyersmann and Hartwig, 1992).
Fig. 5. Some genes of proliferation control modulated by nickel.
6. Nickel Nickel has been extensively studied with respect of gene induction. Nickel has been found to alter the expression of a surprisingly large number of genes (Fig. 5). These include inactivation of senescence genes (Klein and Costa, 1997), inactivation of the antiangionetic thrombospondin gene by induction of the activating transcription factor 1 (Salnikow et al., 1997), silencing of a telomer marker gene (Broday et al., 1999), induction of the hypoxia-regulated gene cap43 (Zhou et al., 1998) and others. Several of these genes are involved in the control of mitogenesis, and these findings provide a hypothesis for the stimulation of cell proliferation in nickel carcinogenesis.
7. Beryllium
Fig. 4. Putative mechanism of induction of growth factor and other genes by cobalt.
Beryllium is the least studied carcinogenic metal with respect of gene modulation. In a study with a stimulated mouse hybrid macrophage cell line, there was no increase in the levels of various transcription factors studied, namely NF-kB, AP1, AP-2, CREB, C/EBP, Sp-1, Egr-1, Ets, NF-Y, and Oct-1 (Hamada et al., 2000). Amongst these are several factors, which are activated by other carcinogenic metal ions in various cell types.
D. Beyersmann / Toxicology Letters 127 (2002) 63–68
67
Hence, the mechanisms by which beryllium induces tumors are still obscure.
magnitude of the present limit values for the working environment which are still in a range that allows for an intolerably high tumor risk.
8. Conclusions and perspectives
References
There is no unifying mechanism in metal carcinogenesis. Individual metals exert differential cellular effects according to their specific physicochemical properties and corresponding interactions with biomolecules. In spite of this caveat, there are striking similarities in the genetic effects of metals. With the exception of chromium(VI), carcinogenic metals are only weak mutagens, if at all. Arsenic, cadmium, cobalt and nickel efficiently inhibit the repair of DNA damage. Hence these metals are assumed to enhance the extent of tumor initiation caused by other agents. Ions of the same metals are active inducers of genes that are known to play a role in the control of cell proliferation. Therefore these metals are postulated to increase the rate of cell growth of initiated cells and act as tumor promoters, too. Both inhibition of DNA repair and stimulation of proliferation may synergistically favor neoplastic growth. A pertinent issue is the question of specificity of metal effects on gene regulation. A priori it cannot be assumed that these effects are part of the physiological signaling network by which cells control growth and differentiation. However, if the genetic alterations are evoked by low, subtoxic concentrations of the metal ions, they may be relevant as relatively sensitive effects of the respective metals. Remarkably, toxic metals also induce genes coding for detoxifying and cytoprotective proteins. Besides differences in cellular uptake and distribution, the balance between detoxifying and deregulating effects may explain the large variations in organ sensitivity to carcinogenic metal compounds. The above-discussed mechanisms are mechanisms involving interactions of metal ions with target proteins, which then indirectly affect the genome. Hence at least theoretically, safe threshold values can be envisaged. Because some of the carcinogenic metal compounds exert their effect at low concentrations in vitro and low exposures in vivo, physiologically based threshold values cannot be in the
Benters, J., Schaefer, T., Beyersmann, D., Hechtenberg, S., 1996. Agonist-stimulated calcium transients are affected differentially by cadmium and nickel. Cell Calcium 20, 441 – 446. Beyersmann, D., Hartwig, A., 1992. The genetic toxicology of cobalt. Toxicol. Appl. Pharmacol. 115, 137 – 145. Beyersmann, D., Hechtenberg, S., 1997. Cadmium, gene regulation, and cellular signalling in mammalian cells. Toxicol. Appl. Pharmacol. 144, 247 – 261. Broday, L., Cai, J., Costa, M., 1999. Nickel enhances telomeric silencing in Saccharomyces cere6isiae. Mutat. Res. 440, 121 – 130. Cavigelli, M., Li, W.W., Lin, A., Su, B., Yoshioka, K., Karin, M., 1996. The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J. 15, 6269 – 6279. De Flora, S., Wetterhahn, K.W., 1989. Mechanisms of chromium metabolism and genotoxicity. Life Chem. Rep. 7, 169 – 244. Ding, W., Templeton, D.M., 2000. Activation of parallel mitogen-activated protein kinase cascades and induction of c-fos by cadmium. Toxicol. Appl. Pharmacol. 162, 93 – 99. El-Sabban, M.E., Nasr, R., Dbaibo, G., Hermina, O., Abboushi, N., Quignon, F., Ameisen, J.C., Bex, F., de The´ , H., Bazarbachi, A., 2000. Arsenic-interferon-a-triggered apoptosis in HTLV-I transformed cells is associated with Tax down-regulation and reversal of NF-kB activation. Blood 96, 2849 – 2855. Gleadle, J.M., Ebert, B.L., Firth, J.D., Ratcliffe, P.J., 1995. Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. Am. J. Physiol. 268, C1362 – C1368. Hamada, H., Sawyer, R.T., Kittle, L.A., Newman, L.S., 2000. Beryllium-stimulation does not activate transcription factors in a mouse hybrid macrophage cell line. Toxicology, 143, 249 – 261. Hartwig, A., 1995. Current aspects in metal genotoxicity. Biometals 8, 3 – 11. Kaltreicher, R.C., Davis, A.M., Lariviere, J.P., Hamilton, J.W., 2001. Arsenic alters the function of the glucocorticoid receptor as a transcription factor. Environ. Health Perspect. 109, 245 – 251. Kapahi, P., Takahashi, T., Natoli, G., Adams, S.R., Chen, Y., Tsien, R.Y., Karin, M., 2000. Inhibition of NF-kB activation by arsenite through reaction with a critical cysteine in the activation loop of I-kB kinase. J. Biol. Chem. 275, 36062 – 36066. Klein, C.B., Costa, M., 1997. DNA methylation, heterochromatin and epigenetic carcinogens. Mutat. Res. 386, 163 – 180.
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Nicotera, P., Zhivotosky, B., Orrenius, S., 1994. Nuclear calcium transport and the role of calcium in apoptosis. Cell Calcium 16, 279 – 288. Porwol, T., Ehleben, W., Zierold, K., Fandrey, J., Acker, H., 1998. The influence of nickel and cobalt on putative members of the oxygen-sensing pathway of erythropoietin-producing HepG2 cells. Eur. J. Biochem. 256, 16 –23. Roussel, R.R., Barchowsky, A., 2000. Arsenic inhibits NF-kBmediated gene transcription by blocking I-kB kinase activity and I-kBa phosphorylation and degradation. Arch. Biochem. Biophys. 377, 204 –212. Seitz, C.S., Lin, Q., Deng, H., Khavari, P.A., 1998. Alterations in NF-kB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-kB. Proc. Natl. Acad. Sci. USA 95, 2307 –2312. Salnikow, K., Wang, S., Costa, M., 1997. Induction of activating transcription factor 1 by nickel and its role as a negative regulator of thrombospondin I gene expression. Cancer Res. 57, 5060 –5066. Simeonova, P.P., Wang, S., Toriuma, W., Kommineni, V., Matheson, J., Unimye, N., Kayama, F., Harki, D., Ding, M., Vallyathan, V., Luster, M.I., 2000. Arsenic mediates
cell proliferation and gene expression in the bladder epithelium: association with activating protein-1 transactivation. Cancer Res. 60, 3445 – 3453. The´ venod, F., Jones, S.W., 1992. Cadmium block of calcium current in frog sympathetic neurons. Biophys. J. 63, 162 – 168. Tully, D.B., Collins, B.J., Overstreet, J.D., Smith, C.S., Dinse, G.E., Mumtaz, M.M., Chapin, R.E., 2000. Effects of arsenic, cadmium, chromium, and lead on gene expression regulated by a battery of 13 different promoters in recombinant HepG2 cells. Toxicol. Appl. Pharmacol. 168, 79 – 90. Wang, Z., Templeton, D.M., 1998. Induction of c-fos protooncogene in mesangial cells by cadmium. J. Biol. Chem. 273, 73 – 79. Ye, J., Zhang, X., Young, H.A., Mao, Y., Shi, X., 1995. Chromium(VI)-induced nuclear factor-kB activation in intact cells via free radical reactions. Carcinogenesis 16, 2401 – 2405. Zhou, D., Salnikow, K., Costa, M., 1998. Cap43, a novel gene specifically induced by Ni2 + compounds. Cancer Res. 58, 2182 – 2189.
Effects of carcinogenic metals on gene expression Detmar Beyersmann * Department of Biology and Chemistry, Uni6ersity of Bremen, D-28334 Bremen, Germany
Abstract Six metals and/or their compounds have been recognized as carcinogens: arsenic, beryllium, cadmium, chromium, cobalt and nickel. With the exception of arsenic, the main rote of exposure is inhalation and the main target organ is the lung. Arsenic is exceptional because it also produces tumors of skin and lung after oral uptake. With the exception of hexavalent chromium, carcinogenic metals are weak mutagens, if at all, and their mechanisms of carcinogenicity are still far from clear. A general feature of arsenic, cadmium, cobalt and nickel is their property to enhance the mutagenicity and carcinogenicity of directly acting genotoxic agents. These properties can be interpreted in terms of the ability of these metals to inhibit the repair of damaged DNA. However, because carcinogenic metals cause tumor development in experimental animals even under exclusion of further carcinogens, other mechanisms have to be envisaged, too. Evidence will be discussed that carcinogenic metal compounds alter patterns of gene expression leading to stimulated cell proliferation, either by activation of early genes (proto-oncogenes) or by interference with genes downregulating cell growth. Special reference will be devoted to the effects of cadmium and arsenic on gene expression, which have been studied extensively. Possible implications for occupational safety and health will be discussed. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Carcinogenic metals; Beryllium; Cadmium; Chromium; Arsenic; Nickel; Cobalt
1. Introduction Six metals and or their compounds have been recognized as human carcinogens, i.e. arsenic, beryllium, cadmium, cobalt, nickel and hexavalent chromium by international agencies (International Agency for Research on Cancer, European Union). With the exception of arsenic, the main route of human exposure is inhalation and the main target organ is the lung. In this respect, * Fax: + 49-421-218-7433. E-mail address: [email protected] (D. Beyersmann).
arsenic is exceptional because it produces skin and lung tumors after oral uptake. With the exception of hexavalent chromium [Cr(VI)], carcinogenic metals are weak mutagens, if at all. Cr(VI) is exceptional because it directly reacts with biological material to produce reactive oxygen species which are able to cause DNA damage and gene mutation (De Flora and Wetterhahn, 1989). The mechanisms of action of carcinogenic metals are still far from being elucidated completely. A general feature of arsenic, cadmium, cobalt and nickel is their property to enhance the mutagenicity and carcinogenicity of directly acting
0378-4274/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 4 8 4 - 2
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D. Beyersmann / Toxicology Letters 127 (2002) 63–68
genotoxic agents. Combined with ultraviolet or ionizing irradiation or other DNA damaging agents, these metals are comutagenic and cocarcinogenic (Hartwig, 1995). A generally applicable mechanism of carcinogenicity of arsenic, cadmium, cobalt and nickel seems to be the inhibition of DNA repair enzymes and the consequent enhancement of DNA damage originally caused by other agents or raised spontaneously. However, because compounds of these metals cause tumors in animals under exclusion of further carcinogens, other mechanisms of transformation to malignant growth have to be envisaged, too. Many carcinogenic metal compounds alter patterns of gene expression. Some of these changes lead to deregulated cell proliferation, either by activation of early genes or by interference with genes downregulating cell growth and senescence. Expression of genes may be modified by interference of the metal ions or their reactive metabolites (e.g. reactive oxygen species) with elements of signal transduction cascades such as second messengers, protein kinases and phosphatases or transcription factors. The classical experimental approach investigates the expression of genes of interest by mRNA and or protein analyses whereas a more recent approach assays changes in
Fig. 1. Effects of arsenic on the expression of selected genes.
a multitude of gene activities by DNA array techniques.
2. Arsenic Genetic effects of arsenic are summarized in Fig. 1. Arsenite activated all major mitogen-activated protein kinase pathways in various mammalian cell lines, which is explained by inhibition of the corresponding protein phosphatases (Cavigelli et al., 1996). Arsenite caused enhanced binding of the mitogenic transcription factor AP-1 to DNA, and it activated the expression of the early genes c-fos, c-myc and egr-1, and of the stress genes gadd153 and gadd45 (Simeonova et al., 2000). The activation of transcription factor AP-1 and the induction of some early genes supports the hypothesis that arsenic promotes neoplastic growth through stimulation of cell proliferation. In another study, effects of metals on gene expression regulated by 13 different promoters in a recombinant cell line were investigated (Tully et al., 2000). Arsenate was found to activate the promoters for MTIIA, GSTYa, HSP70, Fos, XRE, NF-kB, GADD153, p53, and CRE. This list includes several genes coding for cytoprotective proteins, namely metallothionein, glutathione-S-transferase, and some stress proteins. Arsenic also interfered with the transcriptional activity of glucocorticoid receptor complexes, thereby decreasing the expression of genes that downregulate cell proliferation in favor of differentiation (Kaltreicher et al., 2001). Other authors observed that arsenite inhibited the activation of the transcription factor NF-kB and the transcription of genes mediated by this factor (El-Sabban et al., 2000; Roussel and Barchowsky, 2000). This effect was found to be caused by an inhibition of I-kB kinase, an enzyme required for the phosphorylation and degradation of the inhibitor I-kB (Kapahi et al., 2000). Because the dominant effect of NF-kB seems to be growth inhibitory (Seitz et al., 1998), the prevention of NF-kB activation by arsenite points to a further link to the stimulation of cell proliferation in arsenic-induced carcinogenesis.
D. Beyersmann / Toxicology Letters 127 (2002) 63–68
Fig. 2. Examples for genes induced by cadmium.
65
enod and Jones, 1992; Nicotera et al., 1994; Benters et al., 1996) and to cause a sustained activation of mitogen-activated protein kinases that correlated with the induction of c-fos (Wang and Templeton, 1998). The latter effect was specific for cadmium, because six other metal ions tested were inactive (Ding and Templeton, 2000). The activation of mitogenic signaling pathways offers an attractive hypothesis of cadmium carcinogenesis, which in synergy with the inhibition of DNA repair and in antagonism with the stimulation of cytoprotective mechanisms by cadmium, may explain the complex organ specifity in cadmium carcinogenesis.
3. Cadmium 4. Chromium Cadmium is the toxic metal most extensively studied with respect of gene expression (reviewed by Beyersmann and Hechtenberg, 1997). Genetic effects of this metal are summarized in Fig. 2. Similar to arsenic, cadmium induces at least two types of genes: (1) genes coding for detoxifying and other cytoprotective proteins, i.e. metallothioneins, enzymes of glutathione synthesis, heat shock (stress) proteins, zinc transporter proteins; and (2) early genes resp. proto-oncogenes related to cell proliferation control (c-fos, c-jun, c-myc, egr-1). The concentrations of cadmium required for the induction of metallothionein and protooncogenes are remarkably low in the subtoxic range, i.e. a few mM. At variance, stress (heat shock) proteins are induced by cytotoxic cadmium concentrations, i.e. 10– 30 mM, depending on the cell system. The induction processes of metallothioneins and proto-oncogenes are relatively specific for cadmium. This metal is the strongest inducer of metallothionein promoters. Amongst metal ions, cadmium as well as arsenite is a relatively specific inducer of early genes at low concentrations, whereas the stress response seems to be the consequence of more severe cytotoxic damage by elevated cadmium concentrations. The induction of early genes by cadmium is mediated by interference of its ions with cellular signal transduction mechanisms. Cadmium ions have been shown to interact with cellular calcium homeostasis (The´ v-
Chromate(VI) is the only carcinogenic metal species that directly generates reactive oxygen species by interaction with cellular reductants. Hydroxyl radicals, when generated in proximity to DNA, cause DNA strand breaks and oxidized bases (De Flora and Wetterhahn, 1989). Besides causing direct gene mutations, Cr(VI)-evoked formation of OH radicals has been shown to activate nuclear factor-kB which may stimulate inflammatory processes (Ye et al., 1995). In a commercial DNA array test system employing human hepatoma cells, chromate(VI) at low concentrations of 5–10 mM induced the promoters for c-Fos, HSP70, GADD45, NF-kB, p53, XRE and CRE (Tully et al., 2000). A model for the dual cellular effects of chromate(VI) is depicted in Fig. 3.
5. Cobalt At variance, cobalt(II) does not cause direct oxidative stress, but may undergo Fenton-type redox chemistry with hydrogen peroxide (Fig. 4). The resulting decrease in cellular hydrogen peroxide is believed to affect an oxygen-sensing mechanism regulating the hypoxic reaction of human hepatoma cells (Porwol et al., 1998). This leads to the activation of the hypoxia-inducible factor, a transcription factor which induces the expression
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Fig. 3. Mechanisms of DNA damage and gene induction by chromate(VI).
of various genes including those coding for erythropoiesis but also for several growth factors for angiogenesis (Gleadle et al., 1995). The reactive oxygen species generated from hydrogen peroxide may cause DNA damage and mutation, but the mutagenicity of cobalt is very weak (reviewed by Beyersmann and Hartwig, 1992).
Fig. 5. Some genes of proliferation control modulated by nickel.
6. Nickel Nickel has been extensively studied with respect of gene induction. Nickel has been found to alter the expression of a surprisingly large number of genes (Fig. 5). These include inactivation of senescence genes (Klein and Costa, 1997), inactivation of the antiangionetic thrombospondin gene by induction of the activating transcription factor 1 (Salnikow et al., 1997), silencing of a telomer marker gene (Broday et al., 1999), induction of the hypoxia-regulated gene cap43 (Zhou et al., 1998) and others. Several of these genes are involved in the control of mitogenesis, and these findings provide a hypothesis for the stimulation of cell proliferation in nickel carcinogenesis.
7. Beryllium
Fig. 4. Putative mechanism of induction of growth factor and other genes by cobalt.
Beryllium is the least studied carcinogenic metal with respect of gene modulation. In a study with a stimulated mouse hybrid macrophage cell line, there was no increase in the levels of various transcription factors studied, namely NF-kB, AP1, AP-2, CREB, C/EBP, Sp-1, Egr-1, Ets, NF-Y, and Oct-1 (Hamada et al., 2000). Amongst these are several factors, which are activated by other carcinogenic metal ions in various cell types.
D. Beyersmann / Toxicology Letters 127 (2002) 63–68
67
Hence, the mechanisms by which beryllium induces tumors are still obscure.
magnitude of the present limit values for the working environment which are still in a range that allows for an intolerably high tumor risk.
8. Conclusions and perspectives
References
There is no unifying mechanism in metal carcinogenesis. Individual metals exert differential cellular effects according to their specific physicochemical properties and corresponding interactions with biomolecules. In spite of this caveat, there are striking similarities in the genetic effects of metals. With the exception of chromium(VI), carcinogenic metals are only weak mutagens, if at all. Arsenic, cadmium, cobalt and nickel efficiently inhibit the repair of DNA damage. Hence these metals are assumed to enhance the extent of tumor initiation caused by other agents. Ions of the same metals are active inducers of genes that are known to play a role in the control of cell proliferation. Therefore these metals are postulated to increase the rate of cell growth of initiated cells and act as tumor promoters, too. Both inhibition of DNA repair and stimulation of proliferation may synergistically favor neoplastic growth. A pertinent issue is the question of specificity of metal effects on gene regulation. A priori it cannot be assumed that these effects are part of the physiological signaling network by which cells control growth and differentiation. However, if the genetic alterations are evoked by low, subtoxic concentrations of the metal ions, they may be relevant as relatively sensitive effects of the respective metals. Remarkably, toxic metals also induce genes coding for detoxifying and cytoprotective proteins. Besides differences in cellular uptake and distribution, the balance between detoxifying and deregulating effects may explain the large variations in organ sensitivity to carcinogenic metal compounds. The above-discussed mechanisms are mechanisms involving interactions of metal ions with target proteins, which then indirectly affect the genome. Hence at least theoretically, safe threshold values can be envisaged. Because some of the carcinogenic metal compounds exert their effect at low concentrations in vitro and low exposures in vivo, physiologically based threshold values cannot be in the
Benters, J., Schaefer, T., Beyersmann, D., Hechtenberg, S., 1996. Agonist-stimulated calcium transients are affected differentially by cadmium and nickel. Cell Calcium 20, 441 – 446. Beyersmann, D., Hartwig, A., 1992. The genetic toxicology of cobalt. Toxicol. Appl. Pharmacol. 115, 137 – 145. Beyersmann, D., Hechtenberg, S., 1997. Cadmium, gene regulation, and cellular signalling in mammalian cells. Toxicol. Appl. Pharmacol. 144, 247 – 261. Broday, L., Cai, J., Costa, M., 1999. Nickel enhances telomeric silencing in Saccharomyces cere6isiae. Mutat. Res. 440, 121 – 130. Cavigelli, M., Li, W.W., Lin, A., Su, B., Yoshioka, K., Karin, M., 1996. The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J. 15, 6269 – 6279. De Flora, S., Wetterhahn, K.W., 1989. Mechanisms of chromium metabolism and genotoxicity. Life Chem. Rep. 7, 169 – 244. Ding, W., Templeton, D.M., 2000. Activation of parallel mitogen-activated protein kinase cascades and induction of c-fos by cadmium. Toxicol. Appl. Pharmacol. 162, 93 – 99. El-Sabban, M.E., Nasr, R., Dbaibo, G., Hermina, O., Abboushi, N., Quignon, F., Ameisen, J.C., Bex, F., de The´ , H., Bazarbachi, A., 2000. Arsenic-interferon-a-triggered apoptosis in HTLV-I transformed cells is associated with Tax down-regulation and reversal of NF-kB activation. Blood 96, 2849 – 2855. Gleadle, J.M., Ebert, B.L., Firth, J.D., Ratcliffe, P.J., 1995. Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. Am. J. Physiol. 268, C1362 – C1368. Hamada, H., Sawyer, R.T., Kittle, L.A., Newman, L.S., 2000. Beryllium-stimulation does not activate transcription factors in a mouse hybrid macrophage cell line. Toxicology, 143, 249 – 261. Hartwig, A., 1995. Current aspects in metal genotoxicity. Biometals 8, 3 – 11. Kaltreicher, R.C., Davis, A.M., Lariviere, J.P., Hamilton, J.W., 2001. Arsenic alters the function of the glucocorticoid receptor as a transcription factor. Environ. Health Perspect. 109, 245 – 251. Kapahi, P., Takahashi, T., Natoli, G., Adams, S.R., Chen, Y., Tsien, R.Y., Karin, M., 2000. Inhibition of NF-kB activation by arsenite through reaction with a critical cysteine in the activation loop of I-kB kinase. J. Biol. Chem. 275, 36062 – 36066. Klein, C.B., Costa, M., 1997. DNA methylation, heterochromatin and epigenetic carcinogens. Mutat. Res. 386, 163 – 180.
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