Defense Mechanisms and the Shape of Dose-Response Relationships
Environmental Health Perspectives 106, Supplement 1, February 1998
Center for Environmental Health and Occupational Medicine, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
Key words: cadmium, carbon tetrachloride, tolerance, hepatotoxicity, metallothionein induction, subcellular distribution, sequestration, MT-transgenic animals, MT-null mice
This paper is based on a presentation at The Third BELLE Conference on Toxicological Defense Mechanisms and the Shape of Dose-Response Relationships held 12-14 November 1996 in Research Triangle Park, NC. Manuscript received at EHP 7 March 1997; accepted 17 July 1997.
This work was supported by National Institutes of Health grants ES-01142 and ES-07079. We thank the following graduate and postdoctoral fellows who contributed to this project: S.T. Cagen, P. Goering, S. Habeebu, M.B. Iszard, and Y.P. Liu. We also thank R.D. Palmiter, K.H.A. Choo, and G.K. Andrews for their collaboration in these studies.
Address correspondence to Dr. C.D. Klaassen, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160-7417. Telephone: (913) 588-7714. Fax: (913) 588-7501. E-mail: firstname.lastname@example.org
Abbreviations used: CCl4, carbon tetrachloride; MT, metallothionen.
MT-I and -II can be induced easily by heavy metals, hormones, inflammation, acute stress, and many chemicals (1). In essence induction of MT has been proposed as an important adaptive mechanism in response to environmental stimuli. Induction of MT protects against metal toxicity (6), acts as a free radical scavenger protecting against oxidative damage (7), and protects against toxicity of alkylating anticancer drugs and other electrophiles (8).
In this paper we demonstrate that induction of MT is an important cellular adaptive mechanism protecting against the toxicity produced by metals such as Cd as well as by organic chemicals such as carbon tetrachloride (CCl4).
There have been several hypotheses proposed to explain Cd-induced tolerance. Originally it was hypothesized that Cd pretreatment alters the organ distribution of Cd, with more Cd distributing to the liver and less to the kidney. However no major differences in the distribution of Cd to various organs have been observed between control and Cd-pretreated animals (12). It was also hypothesized that tolerance to Cd-induced lethality is attributable to increased biliary excretion of Cd, which has not turned out to be true either. Pretreatment of animals with Cd or Zn actually decreased or prevented the biliary excretion of Cd (14,15).
Why do animals die from acute Cd toxicity? It was originally thought that animals died from Cd-induced cardiotoxicity or nephrotoxicity. However we know now that this is not true. In fact we showed that animals exposed to acute high doses of Cd probably die from liver injury (16). The liver accumulates substantial amounts of Cd after both acute and chronic exposure (6,17). Cd produces dose-dependent liver injury in laboratory animals within 10 hr after iv administration (16-19), with congestion, apoptosis, necrosis, and peliosis as major features of injury (16-20). The Cd-induced liver injury is so severe that hepatic failure is believed to be responsible for acute Cd lethality (12,16,18).
Tolerance to acute Cd toxicity is apparently attributable to MT protection against Cd-induced acute hepatotoxicity (10,12,13). Indeed, after treatment of animals with a low dose of Cd, the liver injury caused by a subsequent toxic dose of Cd is markedly reduced (Figure 1). The hepatoprotection is not attributable to a decreased accumulation of Cd in the liver (12). However, the subcellular distribution of Cd is dramatically altered, with more Cd distributing to the cytosol and a significant reduction of Cd in critical organelles such as nucleus, mitochondria, and microsomes (Figure 2). Chromatography of the cytosolic fraction indicates that most of the Cd in Cd-pretreated animals is associated with MT (Figure 3). Thus the protective role of MT in Cd tolerance to hepatic injury is proposed.
Figure 1. The protective effect of CdCl2 pretreatment (2.0 mg/kg Cd sc for 24 hr) against the hepatotoxic effects of the subsequent high doses of Cd challenge (2.0-5.0 mg/kg Cd iv for 10 hr). Liver injury was measured by plasma aspartate AST. The 5.0 mg/kg dose was not used in control rats as preliminary studies indicate a high rate of mortality in this group. Values represent mean±SE of 4 to 6 rats. *Significantly different from controls at p<0.05. Abbreviation: AST, aminotransferase activity. Reproduced from Goering and Klaassen (12); with permission of Academic Press.
Figure 2. Hepatic subcellular distribution of 109CdCl2 2 hr after challenge (3.5 mg/kg Cd iv) following saline or Cd (2.0 mg/kg sc for 24 hr) pretreatment. The cellular pellets were defined as Nuc (600g, 10 min), Mit (10,000g, 10 min), Mic (100,000g, 60 min), and Cyt (100,000g supernatant). Values represent mean±SE of six rats. *Significantly different from controls at p<0.05. Abbreviations: Cyt, cytosol; Mic, microsomes; Mit, mitochondria; Nuc, nuclei. Reproduced from Goering and Klaassen (10), with permission of Academic Press.
Figure 3. Representative gel-filtration elution profiles of 109CdCl2 in the hepatic cytosols 2 hr after challenge (3.5 mg/kg Cd iv) in control or Cd (2.0 mg/kg sc for 24 hr) pretreated rats. Radioactive Cd eluting with retention coefficients (Ve/Vo) of 1.0 to 1.5 and 1.75 to 2.25 are Cd bound to high-molecular-weight proteins and MT, respectively. Reproduced from Goering and Klaassen (11), with permission of Academic Press.
Newborn animals have high concentrations of MT in their livers; thus they are resistant to Cd-induced lethality and hepatotoxicity. For example Cd treatment (4.0 mg/kg) produced a 20-fold increase in serum alanine aminotransferase activity in adult rats, but in newborns (10-day-old rats) 6 mg Cd/kg did not produce liver injury (21).
Recently we demonstrated that MT-I transgenic mice, which have a 10-fold higher concentration of MT in their liver than control mice (22), are resistant to Cd-induced lethality and hepatotoxicity, as evidenced by >90% lower activities of serum alanine aminotransferase and sorbitol dehydrogenase (Figure 4). In contrast MT-null mice have an increased susceptibility to Cd-induced lethality and hepatotoxicity (23-25), and liver injury is more severe in MT-null mice than in corresponding controls (Figure 5). Furthermore Zn pretreatment, which increases hepatic MT 20-fold in control but not in MT-null mice, protects against Cd-induced hepatotoxicity in control but not in MT-null mice (Figure 5), thus supporting our earlier observation that Zn-induced tolerance to Cd is attributable to induction of MT (26).
Figure 4. Serum ALT and SDH activities in control and MT-TG mice 24 hr after injection of a hepatotoxic dose of CdCl2 (3.1 mg/kg Cd iv). Values represent the mean ± SE of 16 to 25 mice. *Significantly different from controls at p<0.05. Abbreviations: ALT, alanine aminotransferase; SDH, sorbitol dehydrogenase; MT-TG, MT-transgenic mouse. Reproduced from Liu et al. (22), with permission of Academic Press.
Figure 5. SDH activities in control and MT-null mice pretreated with saline or Zn (200 µmol/kg, sc2) and subsequently challenged with a hepatotoxic dose of CdCl2 (2.8 mg/kg Cd ip for 16 hr). Values are mean±SE (n=16-24). *Significantly different from control mice at p<0.05. **Significant difference between CdCl2 and Zn+CdCl2 groups at p<0.05. Reproduced from Liu et al. (25), with permission of Williams & Wilkens.
These data indicate that both constitutive and inducible MT are responsible for the detoxication of Cd. Pharmacodynamic tolerance occurs via high-affinity sequestration of the metal within the cell. As a result most of the Cd in cells is bound to MT in the cytosol, with a concomitant reduction of the Cd available to bind/damage critical organelles (6). Using MT-null mice, we find that intracellular MT also plays an important protective role in chronic Cd nephrotoxicity (J Liu et al., in preparation). Binding of metal ions to MT also appears to be the mechanism for the protection against the toxicity of other metals such as mercury, silver, and cisplatin (27,28). Thus, induction of MT is an important adaptive mechanism preventing metal toxicity in animals as well as in humans.
Figure 6. The protective effect of Zn pretreatment (ZnCl2 250 µmol/kg ip) against the hepatotoxicity of carbon tetrachloride (0.3-1.75 ml/kg in corn oil, ip for 24 hr). Liver injury was measured by serum ALT. Values represent mean±SE of 4 to 6 rats. *Significantly different from controls at p<0.05. Reproduced from Goering and Klaassen (30), with permission of Academic Press.
Figure 7. Representative gel-filtration elution profiles of 14C-Cl4 in the hepatic cytosols 90 min after administration of 14C-Cl4 (25 µCi/kg ip) in control or Zn (250 µmol/kg, ip for 24 hr) pretreated rats. Radioactive 14C-Cl4 eluting in fractions 15 to 25 and fractions 45 to 55 are 14C-Cl4 bound to high-molecular-weight proteins and MT, respectively. Reproduced from Goering and Klaassen (30), with permission of Academic Press.
The potential role of MT induction as an adaptive mechanism decreasing CCl4 toxicity is further supported by other studies. First, 24-hr pretreatment with Zn increased hepatic MT and protected against CCl4 toxicity, while 2-hr Zn pretreatment prior to the induction of MT synthesis failed to offer protection (31). Second, mild Zn deficiency interferes with MT synthesis and thus decreases the efficacy of MT induction by turpentine to protect against CCl4 toxicity (32). Third, recent studies showed that MT-null mice are more susceptible than controls to CCl4 hepatotoxicity (33,34), indicating that MT functions as an adaptive mechanism to decrease the toxicity of CCl4.
Evidence also suggests a role for MT in protection aginst oxidative stress. MT can serve as a sacrificial scavenger for hydroxyl radicals in vitro (35) and protect against free radical-induced DNA damage (36-38). MT can also assume the function of superoxide dismutase in yeast (39) and protect against lipid peroxidation in erythrocyte ghosts produced by xanthine oxidase-derived superoxide anion and hydrogen peroxide (40). Hepatocytes from MT-null mice are more sensitive than control cells to oxidative damage produced by t-butylhydroperoxide and paraquat (41,42). MT is induced by oxidative stress-producing chemicals (43) and thus may protect against oxidative damage (7) and the toxicity of alkylating anticancer drugs (8).
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Last Update: March 11, 1998