Metallothionein-I/II null mice are more sensitive than wild-type mice to the hepatotoxic and nephrotoxic effects of chronic oral or injected inorganic arsenicals.

Metallothionein (MT) is a low-molecular-weight, sulfhydryl-rich, metal-binding protein that can protect against the toxicity of cadmium, mercury, and copper. However, the role of MT in arsenic (As)-induced toxicity is less certain. To better define the ability of MT to modify As toxicity, MT-I/II knockout (MT-null) mice and the corresponding wild-type mice (WT) were exposed to arsenite [As(III)] or arsenate [As(V)] either through the drinking water for 48 weeks, or through repeated sc injections (5 days/week) for 15 weeks. Chronic As exposure increased tissue MT concentrations (2-5-fold) in the WT but not in MT-null mice. Arsenic by both routes produced damage to the liver (fatty infiltration, inflammation, and focal necrosis) and kidney (tubular cell vacuolization, inflammatory cell infiltration, and interstitial fibrosis) in both MT-null and WT mice. However, in MT-null mice, the pathological lesions were more frequent and severe when compared to WT mice. This was confirmed biochemically, in that, at the higher oral doses of As, blood urea nitrogen (BUN) levels were increased more in MT-null mice (60%) than in WT mice (30%). Chronic As exposures produced 2-10 fold elevation of serum interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha levels, with greater increases seen by repeated injections than by oral exposure, and again, MT-null mice had higher serum cytokines than WT mice after As exposure. Repeated As injections also decreased hepatic glutathione (GSH) by 35%, but GSH-peroxidase and GSH-reductase were minimally affected. MT-null mice were more sensitive than WT mice to the effect of GSH depletion by As(V). Hepatic caspase-3 activity was increased (2-3-fold) in both WT and MT-null mice, indicative of apoptotic cell death. In summary, chronic inorganic As exposure produced injuries to multiple organs, and MT-null mice are generally more susceptible than WT mice to As-induced toxicity regardless of route of exposure, suggesting that MT could be a cellular factor in protecting against chronic As toxicity.

Toxic and essential metal interactions.

Cadmium, lead, mercury, and aluminum are toxic metals that may interact metabolically with nutritionally essential metals. Iron deficiency increases absorption of cadmium, lead, and aluminum. Lead interacts with calcium in the nervous system to impair cognitive development. Cadmium and aluminum interact with calcium in the skeletal system to produce osteodystrophies. Lead replaces zinc on heme enzymes and cadmium replaces zinc on metallothionein. Selenium protects from mercury and methylmercury toxicity. Aluminum interacts with calcium in bone and kidneys, resulting in aluminum osteodystrophy. Calcium deficiency along with low dietary magnesium may contribute to aluminum-induced degenerative nervous disease.

Prenatal care and infant lead exposure.

OBJECTIVE: The objective of the study was to determine the relation between prenatal care of mothers and blood lead concentrations in their offspring in the first year of life. METHODS: A retrospective survey was conducted of 200 predominantly black infants between the ages of 6 and 22 months (mean age, 13.4 months). The infants had been screened for the first time since birth at the Charleston County (South Carolina) Health Department. They resided in a neighborhood with the highest prevalence of lead poisoning in Charleston. Prenatal care use data were obtained after matching birth records with lead-screening records. RESULTS: Seventy-three infants (37%) had blood lead levels 0.48 micromol/L (> or = 10 microg/dl) or higher. Adequacy of prenatal care, defined by the Modified Kessner Index, showed 11% with intensive care (26% of these with high lead levels), 39% with adequate care (35% high blood lead levels), 35% with intermediate care (40% with high blood lead levels), 13% with inadequate care (42% with high blood lead levels), and 2% with no prenatal care (25% with high blood lead levels). With the exception of the small group with no prenatal care (n = 4), the proportion of infants with a high blood lead level was inversely proportional to the level of care. The logistic regression model that best fit the data included age at screen for lead and birth weight. Low birth weight babies (<2500 gm) were more likely to have a high blood lead level at primary screen than babies who were heavier at birth (odds ratio, 2.60; p = 0.04), and the older the baby at screening, the greater the likelihood of a high blood lead level (odds ratio, 1.23; p = 0.01). There was a trend for black infants to have a high blood lead level more often than white infants (odds ratio, 3.05; p = 0.06). CONCLUSIONS: Less than adequate use of prenatal care may reflect an increase in risk factors contributing to lead exposure in infancy. Low birth weight also was related to high blood lead levels. Further studies are required to differentiate among several hypotheses for this effect. Intrauterine lead exposure, which is known to reduce birth weight, may contribute to measured blood lead levels at first screen. Alternatively, low birth weight may increase lead absorption and retention in infancy or may increase risk of lead exposure.

Results of lead research: prenatal exposure and neurological consequences.

The history of advances in the understanding of the toxic effects of lead over the past 20 years is an outstanding example of how knowledge learned from research can impact public health. Measures that have had the greatest impact on reducing exposure to lead are reduction of lead from gasoline, elimination of lead solder from canned food, removal of lead from paint, and abatement of housing containing lead-based paint. Nevertheless, continuing factors that enhance risk to lead exposure, particularly during fetal life, are low socioeconomic status, old housing with lead-containing paint, and less than ideal nutrition, particularly low dietary intake of calcium, iron, and zinc. Prenatal exposure may result from endogenous sources such as lead in the maternal skeletal system or maternal exposures from diet and the environment. Experimental studies have shown that the developing nervous system is particularly sensitive to the toxic effects of lead and that a large number of the effects in the nervous system are due to interference of lead with biochemical functions dependent on calcium ions and impairment of neuronal connections dependent on dendritic pruning. There is need for more study to determine whether these effects are a continuum of prenatal lead exposure or whether prenatal exposure to lead produces unique effects.

Down-regulation of metallothionein expression in human and murine hepatocellular tumors: association with the tumor-necrotizing and antineoplastic effects of cadmium in mice.

Previously, we found that oral cadmium (Cd) treatment either prevented or substantially reduced N-nitrosodiethylamine (NDEA)-induced tumor formation in B6C3F1 mouse liver or lung regardless of exposure interval and even when the Cd was given well after tumors were formed. Because Cd salts are powerful emetics, oral exposure would probably be impractical in humans. Thus, we studied suppression of NDEA-initiated tumors in male B6C3F1 mice by a single i.v. dose of Cd. NDEA (776 mumol/kg i.p.) was given at time 0 followed by CdCl2 (16 mumol/kg i.v.) 40 weeks later. This dose of Cd had no effect on body weights through the conclusion of the study at 52 weeks. The NDEA-induced increase in hepatic tumor incidence (19 tumor-bearing mice/22 mice at risk, 86%) over control (5/24, 21%) was remarkably reduced by Cd treatment (13/27, 48%, P < or = .05). Multiplicity and size of liver tumors induced by NDEA (2.18 tumors/liver; 31.6 mm3 mean volume) were also substantially reduced by the Cd exposure (0.96 tumors/liver; 17.1 mm3 mean volume). NDEA-induced lung tumor incidence (22/22, 100%) and multiplicity (5.09 tumors/lung) were modestly, but significantly, reduced by Cd treatment (21/27, 78%; 3.89 tumors/lung). Clear evidence of tumor-specific cytotoxicity was observed as Cd treatment induced a necrotizing effect that was localized only within the hepatic tumors. Metallothionein (MT), an inducible metal-binding protein associated with tolerance to many metal including Cd, was not detected immunohistochemically in mouse liver tumors, even those undergoing Cd-induced necrosis, whereas the surrounding normal liver cells expressed high levels of MT after Cd exposure. Likewise, in human hepatocellular carcinomas MT was only poorly or erratically expressed relative to normal tissue. These results indicate that a single, nontoxic dose of Cd dramatically reduces liver tumor burden through tumor cell-specific necrosis due to a down-regulation of MT expression in hepatic tumors of murine origin and furthermore indicate that a similar down-regulation of MT occurs in human hepatocellular carcinomas.

Renal tubular tumors and atypical hyperplasias in B6C3F1 mice exposed to lead acetate during gestation and lactation occur with minimal chronic nephropathy.

Lead is a high-priority hazardous substance in humans and a renal carcinogen in adult rodents. This study assessed the carcinogenic potential and toxicity of gestational and lactational lead exposure in (C57BL/6NCr x C3H/HeN)F1 (hereafter called B6C3F1) mice. Effects of a renal tumor promoter [barbital sodium (BB)] on lead-initiated lesions were also studied. Pregnant female C57BL/6NCr mice (10-15/group) previously bred with C3H/HeN males were given lead acetate (0, 500, 750 and 1000 ppm lead) ad libitum in their drinking water, starting on gestation day 12 and continuing to 4 weeks postpartum. Offspring were then weaned and divided into same-sex groups of 23-25 and observed for a maximum of 112 weeks. Other groups received lead and then continuous BB (500 ppm) ad libitum in their drinking water from weaning onward. In control male offspring (0 lead/0 BB), renal proliferative lesions [(RPLs); defined as atypical tubular hyperplasia or tumor] occurred rarely (1 lesion-bearing mouse/23 mice examined, 4%) and did not include tumors. RPLs increased in a dose-related fashion with lead exposure (500 lead/0 BB, 4/25, 16%; 750 lead/0 BB, 6/25, 24%; 1000 lead/0 BB, 12/25, 48%) in male offspring and were often multiple. All lead-treated groups had renal tumors, including carcinoma, but these were most common at the highest dose (1000 lead/0 BB, 5/25). Lead-induced renal tumors arose in the absence of the extensive chronic nephropathy and lead inclusion bodies typically seen with lead carcinogenesis in rodents exposed chronically as adults. Postnatal BB exposure had no effect on RPL incidence (e.g., 1000 lead/500 BB, 8/25, 32%). Lead-treated female offspring also developed RPLs, including adenoma and carcinoma, but at a much lower rate than males. Thus, short-term lead exposure during the gestational/lactational period has carcinogenic potential in the mouse kidney.

Nutrition and metal toxicity.

Lead, cadmium, and mercury are toxic metals that are not essential for nutrition. However, the toxic effects of these metals may be mediated or enhanced by interactions or deficiencies of nutritionally essential metals. Lead competes with calcium, inhibiting the release of neurotransmitters, and interferes with the regulation of cell metabolism by binding to second-messenger calcium receptors, blocking calcium transport by calcium channels and calcium-sodium ATP pumps, and by competing for calcium-binding protein sites and uptake by mitochondria. Dietary deficiencies of calcium, iron, and zinc enhance the effects of lead on cognitive and behavioral development. Iron deficiency increases the gastrointestinal absorption of cadmium, and cadmium competes with zinc for binding sites on metallothionein, which is important in the storage and transport of zinc during development. Selenium protects from mercury and methyl mercury toxicity by preventing damage from free radicals or by forming inactive selenium mercury complexes.

Immunomodulation by metals.

A symposium entitled Immunomodulation by Metals was held at the 32nd Annual Meeting of the Society of Toxicology (SOT) in New Orleans, Louisiana. The symposium was co-sponsored by the Immunotoxicology and Metals Specialty Sections of SOT and was designed to describe the types of adverse immunological reactions which occur in response to environmental and/or occupational exposure to metals. Epidemiological evidence and underlying mechanisms responsible for the observed alterations were also discussed. The following is a summary of each of the individual presentations.

Nephrotoxicity in rats following liver transplantation from cadmium-exposed rats.

Although kidney is considered as the critical organ for cadmium (Cd) toxicity, little is known about the transport of Cd to kidney after chronic exposure. In order to study this transfer, male Lewis rats (150-200 g) were given eight injections (sc) of CdCl2 (3 mg Cd/kg) over 2 weeks which resulted in increases of tissue Cd and metallothionein (MT) concentrations (223 and 1850 micrograms/g, respectively, in the liver and 118 and 873 micrograms/g, respectively, in the kidney). Livers from Cd-injected rats were transplanted to age-matched control healthy Lewis rats and the recipient rats were killed at 2 to 47 days after transplantation. The levels of Cd and MT in the liver of recipient rats were decreased (106 and 1503 micrograms/g, respectively) with time after surgery. On the other hand, renal Cd and MT levels were markedly increased (195 and 1468 micrograms/g, respectively) and most of the Cd in the kidney was bound to MT. About 100 ng/ml of Cd and MT were detected in the plasma of recipient rats by ELISA. There was some periportal fibrosis in the liver due to transplant procedure which did not anastomose hepatic arteries. There was an increase in blood urea nitrogen levels in rats transplanted with Cd-containing liver. In addition, both necrosis and inflammation were observed in the epithelial cells in the proximal tubules in the kidney which typically occurs in chronic Cd toxicity. These results suggest that the major source of renal Cd in chronic Cd exposure may be derived from hepatic Cd which is transported in the form of Cd-MT in blood plasma.

Further evidence of the tumor-suppressive effects of cadmium in the B6C3F1 mouse liver and lung: late stage vulnerability of tumors to cadmium and the role of metallothionein.

Previously, we studied the ability of cadmium to initiate or promote tumors in B6C3F1 mice and, contrary to expectation, found that cadmium inhibited development of N-nitrosodiethylamine (NDEA)-initiated and sodium barbital-promoted liver tumors. In this study, the time course of cadmium inhibition of NDEA-initiated tumor formation was studied. A single dose of NDEA (90 mg/kg i.p.) was given at 5 weeks of age (time 0) followed by cadmium (1000 ppm) in drinking water from 2 to 48, 4 to 48, 8 to 48, 16 to 48 and 32 to 48 weeks. The study ended at 48 weeks. NDEA-induced elevations in liver tumor incidence (22 tumor-bearing mice/25 total) over control (5/25) were prevented by cadmium regardless of the period of administration (NDEA + cadmium: 2-48 weeks, 2/25; 4-48 weeks, 1/25; 8-48 weeks, 1/25; 16-48 weeks, 2/25; 32-48 weeks, 6/24). Cadmium alone (2-48 weeks) eliminated (0/25) spontaneously occurring liver tumors (5/25). NDEA-induced lung tumor incidence (25/25) and multiplicity (7.28 tumors/lung) were also reduced by cadmium (maximal decreases 28% and 80%, respectively). Some evidence of a specific deficiency of metallothionein in tumor cells was seen immunohistologically in NDEA-induced hepatic lesions and pulmonary lesions. These results indicate that cadmium prevents or reduces tumor formation in the B6C3F1 mouse liver and lung regardless of the exposure interval and apparently by cell-specific cytotoxicity. Auxiliary studies indicated that in mice bearing multiple liver foci resulting from NDEA treatment there was a marked reduction in basal metallothionein levels and in response to zinc induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnancy-associated changes in plasma metallothionein concentration and renal cadmium accumulation in rats.

Pregnancy-associated changes in metallothionein (MT) concentrations in blood plasma were examined using a competitive enzyme-linked immunosorbent assay with a rabbit polyclonal antibody to rat liver MT. Plasma MT of pregnant rats significantly increased after 8 days of gestation and remained high during pregnancy and for 7 days after delivery. Gel filtration showed that both Cu and Zn were associated with the plasma MT of pregnant rats. These results suggest that plasma MT may play a role in the transport of essential metals such as Cu and Zn to fetus during pregnancy, but the source of plasma MT is unknown. Injections of cadmium chloride and cadmium-metallothionein to pregnant rats further increased the plasma MT concentrations. After injection of CdCl2, both MT and Cd concentrations in the liver of the pregnant rats were significantly lower than those of the nonpregnant rats, whereas renal Cd and MT levels were higher in pregnant rats. This increased accumulation of Cd in the kidney of pregnant rats may be related to the increase of plasma MT during pregnancy. About 5% of 6 pg of in vitro added Cd (20 pg/ml) was bound to the MT in the plasma. Therefore, Cd may be transported by the circulating MT to the kidney, leading to an increase in renal accumulation of Cd in pregnant rats.

Lead toxicity: current concerns.

Over the 20-year period since the first issue of Environmental Health Perspectives was published, there has been considerable progress in the understanding of the potential toxicity of exposure to lead. Many of these advances have been reviewed in published symposia, conferences, and review papers in EHP. This brief review identifies major advances as well as a number of current concerns that present opportunities for prevention and intervention strategies. The major scientific advance has been the demonstration that blood lead (PbB) levels of 10-15 micrograms/dL in newborn and very young infants result in cognitive and behavioral deficits. Further support for this observation is being obtained by prospective or longitudinal studies presently in progress. The mechanism(s) for the central nervous system effects of lead is unclear but involve lead interactions within calcium-mediated intracellular messenger systems and neurotransmission. Effects of low-level lead exposure on blood pressure, particularly in adult men, may be related to the effect of lead on calcium-mediated control of vascular smooth muscle contraction and on the renin-angiotensin system. Reproductive effects of lead have long been suspected, but low-level effects have not been well studied. Whether lead is a carcinogen or its association with renal adenocarcinoma is a consequence of cystic nephropathy is uncertain. Major risk factors for lead toxicity in children in the United States include nutrition, particularly deficiencies of essential metals, calcium, iron, and zinc, and housing and socioeconomic status. A goal for the year 2000 is to reduce prevalence of blood lead levels exceeding 15 micrograms/dL.

Nephrotoxicity of repeated injections of cadmium-metallothionein in rats.

Cadmium-metallothionein (Cd-MT) may have a role in the pathogenesis and irreversibility of Cd nephrotoxicity. In the present study, rats were injected with 0.3 mg Cd/kg body wt per week as Cd-MT for 5 consecutive weeks and a group of rats (n = 3) was killed 24 hr after each injection. A group of three rats was kept for an additional week after the 5 weeks of Cd-MT injection for recovery. After the first injection, urinary Cd and protein levels and kidney/body wt ratio were increased. The electrophoretic pattern of urinary protein showed increased excretion of low-molecular-weight proteins, especially after the first injection of Cd-MT. Tubular cell necrosis occurred after the first week with renal Cd levels of only 10 micrograms/g and gradually progressed to severe necrosis with inflammation in 3 weeks and then to interstitial fibrosis in 5 weeks. The levels of Cd and MT in kidney increased with repeated injection of Cd-MT, but renal Cd was about 40 micrograms/g after 5 weeks of injection. Urinary Cd and MT levels progressively increased during the Cd exposure period, but returned to pretreatment levels during the sixth week (recovery period). Renal cell necrosis and inflammation were absent at the sixth week, but interstitial fibrosis persisted. This study indicates that nephrotoxicity of Cd in this model is related to urinary excretion of Cd-MT and that renal cell injury may be independent of Cd in the renal cortex. Nephrotoxicity occurs at levels much lower than the proposed critical concentration for Cd (200 micrograms Cd/g) following long-term exposure to CdCl2. However, in the absence of continued Cd exposure from liver or circulation, the Cd-MT-induced renal damage is reversible.

Cellular localization of metallothionein in human term placenta.

Cellular localization of metallothionein (MT) in placenta may provide information on its function as a metal binding protein. Rabbit antibodies to rat liver MT cross-reacted with human MT and were used to localize MT in human term placenta by avidin-biotin peroxidase technique. Serial sections (5 microns) were cut from paraffin-embedded placentae obtained at term from five normal women and incubated with rabbit antibodies to MT. Normal rabbit serum was used as a negative control. The slides were incubated with biotinylated swine anti-rabbit IgG (linking antibody) then with avidin-biotin horseradish peroxidase complex and developed with diaminobenzidine in hydrogen peroxide (0.03 per cent) substrate. The optimum staining of MT was obtained at a 1:800 antibody dilution. MT was identified in fetal amniotic cells, syncytial trophoblasts and villous interstitial cells, and in maternal decidual cells. The presence of MT at specific cellular sites suggests that it may regulate the transplacental transport of metals such as zinc, copper and cadmium. Since the level of cadmium is lower and that of zinc and copper higher in fetal than in maternal blood, this may suggest that placental MT may restrict cadmium while enhancing zinc and copper transport.

Immunohistochemical evidence of high concentrations of metallothionein in pancreatic hepatocytes induced by cadmium in rats.

A recent study from our laboratory has shown that cadmium, a toxic heavy metal, is one of the most effective agents known for inducing hepatocytic transdifferentiation of the rat pancreas. With repeated injections of cadmium, the incidence of rats with pancreatic hepatocytic foci can be as high as 93%. Cadmium is also well known as a very potent inducer of metallothionein, a metal-binding protein that appears to be important in the biologic response to several toxic heavy metals in most tissues, including the pancreas. Therefore, the present study sought to determine if metallothionein was associated with cadmium-induced transdifferentiation of pancreatic cells. Expression of metallothionein was studied immunohistochemically by the peroxidase-antiperoxidase method in tissue sections of the pancreas of rats with pancreatic hepatocytes. High levels of metallothionein were localized primarily within the pancreatic hepatocytes. Surrounding normal pancreatic islet and acinar cells were not immunoreactive. Thus, metallothionein is expressed actively in cells transdifferentiated to hepatocytes by cadmium within the pancreas.

Role of metallothionein in human placenta and rats exposed to cadmium.

A strong positive relationship between zinc and copper and metallothionein (MT) in placentas has been found, but a negative one between cadmium and MT. In rats given cadmium i.p. as the chloride, liver cadmium is lower and kidney cadmium higher than in cadmium-treated non-pregnant rats, suggesting that pregnancy enhances mobilization of cadmium from liver to kidney. The cadmium concentration of digested whole fetuses is not significantly increased in offspring of dams given cadmium i.p. as the chloride or CdMT, but the placental levels of cadmium and MT are increased. The placenta therefore acts as a barrier to maternal-fetal cadmium transfer. The way in which cadmium is retained in the placenta but zinc and copper are transferred to the fetus is not understood, since all are bound to MT. Focal renal tubular necrosis and placental necrosis occur at the same level of cadmium exposure, suggesting a similar threshold to cadmium toxicity for the two organs.