Induction of metallothionein as an adaptive mechanism affecting the magnitude and progression of toxicological injury.

Pretreatment of rats with low doses of Cd produces adaptive tolerance to a subsequent high dose of Cd-induced lethality, thus shifting the dose-response curve to the right. Cd pretreatment of animals also protects against the hepatotoxicity produced by high doses of Cd. This protection is attributable to the 10- to 50-fold induction of hepatic metallothionein (MT) by Cd pretreatment. As a result hepatic subcellular distribution of Cd is significantly altered, with more Cd bound to MT in the cytosol and a concomitant reduction of Cd in other critical organelles. In addition MT-transgenic animals are more resistant, whereas MT-null mice are more sensitive than controls to Cd-induced lethality and hepatotoxicity. This further demonstrates that MT is important in Cd detoxication. Induction of hepatic MT by zinc also protects mice from carbon tetrachloride (CCl4)-induced liver injury, with more 14C-CCl4 bound to MT in the cytosol. MT-null mice are more sensitive to CCl4-induced hepatotoxicity, which supports the hypothesis that induction of MT also plays a protective role for nonmetallic chemicals. These results indicate that MT is a part of cellular adaptive mechanisms affecting the magnitude and progression of toxic insults from metals such as Cd as well as from organic chemicals such as CCl4. ImagesFigure 2Figure 4Figure 5


Introduction
Metallothionein (MT) is a low-molecularweight protein ubiquitous in the animal kingdom (1). MT has an unusual amino acid composition in that it has no aromatic amino acids and one-third of its residues are cysteines. These cysteine residues bind and store metal ions (2). The MT multigene family is composed of at least four isoforms.
MT-I and -II exist in all tissues, are regulated in a coordinate fashion, and appear functionally equivalent (1)(2)(3). Other members of the MT gene family, however, show different patterns of expression: MT-III is found mainly in brain (4) and MT-IV in stratified squamous epithelium (5). MT-Ill and -IV are regulated very differently than MT-I and -II and their significance is not yet understood.
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 (CC14). Induction ofMetallothionein

Protects against Cadmium Toxicity
Cd is an environmental pollutant that is toxic to a number of tissues, including liver, kidney, lung, bone, reproductive organs, and the immune system (6). However, following low doses of Cd exposure, animals become tolerant to a subsequent lethal dose of Cd (9)(10)(11)(12)(13).
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 Cdinduced 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)(17)(18)(19), with congestion, apoptosis, necrosis, and peliosis as major features of injury (16)(17)(18)(19)(20). The Cdinduced 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). T-he hepatoprotection is not attributable to a decreased accumulation of Cd in the liver (12). However, the subcellular distribution Environmental Health Perspectives * Vol 106, Supplement 1 * February 1998   (12); with permission of Academic Press.
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. 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 Cdinduced 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)(24)(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 Representative gel-filtration elution profiles of 109CdC12 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.
Zn-induced tolerance to Cd is attributable to induction of MT (26). 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 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. Induction ofMetallothionein as an Adaptive Mechanism for Nonmetallic Chemicals MT has a high cysteine content (30%) and thus may be similar to glutathione in providing another nucleophilic sink in the cell that can bind electrophiles. MT in the liver is induced by CCl4 administration (29), indicating a possible role for this protein to provide tolerance to CCl4 toxicity. We demonstrated that induction of MT by Zn protects against CC14 hepatotoxicity Environmental Health Perspectives * Vol 106, Supplement * February 1998 offer protection (31). Second, mild Zn deficiency interferes with MT synthesis and thus decreases the efficacy of MT induction by turpentine to protect against CC14 toxicity (32). Third, recent studies showed that MT-null mice are more susceptible than controls to CC14 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)(37)(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).

Summary
MT, a cysteine-rich, metal-binding protein, exists in most tissues and is easily induced by many stimuli. Thus induction of MT is an important cellular adaptive mechanism in response to environmental insults. MT plays an important role in Cd tolerance and Cd-induced hepatotoxicity. MT binds Cd in the hepatic cytosol and renders it inert. MT may also function similarly as glutathione in providing a nucleophilic sink, and binding electrophiles. MT may also act as a free radical scavenger to protect against oxidative damage as an adaptive mechanism in response to nonmetallic toxicants. The functions of MT warrant further investigation.