In vitro human skin irritation test for evaluation of medical device extracts.

The aim of this study was to determine if the EpiDerm™ reconstructed human skin model (MatTek Corp.) could be an acceptable alternative to the ISO 10993-required rabbit skin irritation test for assessing medical device biocompatibility. Eleven medical device polymers were tested. Four extracts were prepared per polymer, two each with saline and sesame oil; half were spiked with two R-38 irritants, lactic acid for saline extracts and heptanoic acid for the sesame oil extracts. Tissue viability was assessed by MTT reduction and the proinflammatory response was assessed by IL-1α release. LOAELs of 2% for lactic acid in saline and 0.7% for heptanoic acid in sesame oil were determined. A cell viability reduction of >50% was indicative of skin irritation. Cells exposed to saline extracts spiked with 3.25% lactic acid had significantly reduced mean cell viabilities (12.6-17.2%). Cells exposed to sesame oil extracts spiked with 1.25% heptanoic acid also exhibited reduced mean cell viabilities (25.5%-41.7%). All spiked cells released substantial amounts of IL-1α (253.5-387.4pg/ml) signifying a proinflammatory response. These results indicate that the EpiDerm™ model may be a suitable in vitro replacement for the assessment of the irritation potential of medical device extracts.

Potential estrogenic and antiestrogenic activity of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and the linear siloxane hexamethyldisiloxane (HMDS) in immature rats using the uterotrophic assay.

The cyclic siloxane octamethylcyclotetrasiloxane (D4) and the linear siloxane hexamethyldisiloxane (HMDS) have numerous industrial and consumer applications and thus have the potential for human exposure. The present study was undertaken to examine potential estrogenic and antiestrogenic activities of D4 and HMDS. To address potential differences in sensitivity between rat strains the study used both Sprague-Dawley (SD) and Fischer 344 (F-344) rats. Estrogenicity of the test compounds was determined by measuring absolute and relative uterine weights in immature rats and by monitoring uterine epithelial cell height. In order to place the data obtained for D4 into perspective relative to strong and weak estrogenic compounds, the response produced by D4 at 0, 10, 50, 100, 250, 500, and 1000 mg/kg/day was compared to responses produced by ethinyl estradiol (EE) (1, 3, 10, or 30 microg/kg/day), diethylstilbestrol dipropionate (DES-DP) (0.5, 1.5, 5, 15 microg/kg/day), and coumestrol (CE) (10, 35, 75, 150 mg/kg/day). Antiestrogenic effects were evaluated by co-administering D4 (500 mg/kg/day) with EE at 1, 3, 10, and 30 microg /kg/day. All compounds were administered in sesame oil at a volume of 5 mL/kg by oral gavage. Beginning on postnatal day 18 (SD) or 21 (F-344) each pup (12 per group) received a single dose of test compound once a day for 4 consecutive days. The pups were euthanized the morning after the last treatment and their uteri removed, weighed, and processed for histological examination. EE and DES-DP produced a significant dose-dependent increase in absolute and relative uterine weights and uterine cell height. The maximum increase in uterine weight following EE exposure was approximately 350% relative to controls in both strains. The weak phytoestrogen CE also produced a dose-related increase in absolute and relative uterine weight and epithelial cell height, but the response occurred over a much higher range of doses. At the highest dose of CE, uterine weight was increased approximately 230% relative to controls. Following exposure to D4, absolute and relative uterine weights and uterine epithelial cell height were statistically significantly increased in both strains of rats at doses above 100 mg/kg/day. In terms of uterine weight, D4 was approximately 0.6 million times less potent than EE or DES-DP in SD pups and 3.8 million times less potent than EE or DES-DP in F-344 pups. The maximal increase in uterine weight, relative to controls, produced by D4 at 1000 mg/kg/day was approximately 160% in SD rats, while the maximum increase produced by D4 in F-344 rats was 86%. D4 co-administered over a wide range of EE doses, resulted in a significant reduction in uterine weight compared to EE alone. HMDS was evaluated in SD rats only. The response produced by HMDS (600 and 1200 mg/kg/day) was compared to EE (3 microg/kg/day). Antiestrogenic effects were evaluated by co-administering HMDS (1200 mg/kg/day) with EE at 3 microg/kg/day. HMDS had no measurable effect on uterine weight under the experimental conditions described here. However, HMDS coadministered with EE did produce a small, but statistically significant reduction in uterine weight compared to EE alone. In conclusion, D4 showed weak estrogenic and antiestrogenic activity that was several orders of magnitude less potent than EE, and many times less potent than the weak phytoestrogen CE.

Repeated inhalation exposure to octamethylcyclotetrasiloxane produces hepatomegaly, transient hepatic hyperplasia, and sustained hypertrophy in female Fischer 344 rats in a manner similar to phenobarbital.

Octamethylcyclotetrasiloxane (D4) has been described as a phenobarbital-like inducer of hepatic enzymes. Phenobarbital (PB) and phenobarbital-like chemicals induce transient hepatic and thyroid hyperplasia and sustained hypertrophy in rats and mice. The extent to which these processes are involved with D4-induced hepatomegaly is not known. The present study has evaluated the effects of repeated inhalation exposure to D4 vapors on hepatic and thyroid cell proliferation and hypertrophy with respect to time and exposure concentration. Female Fischer 344 rats were exposed via whole body inhalation to 0 ppm D4, 700 ppm D4 vapors (6 h/day; 5 days/week), or 0.05% PB in drinking water over a 4-week period. Incorporation of 5'-bromo-2-deoxyuridine (BrdU) and the abundance of proliferating cell nuclear antigen were used as indicators of cell proliferation. Designated animals from each treatment group were euthanized on study days 6, 13, and 27. The effect of D4 exposure concentration on hepatic cell proliferation was evaluated at 0, 7, 30, 70, 150, 300, or 700 ppm. Liver-to-body weight ratios in animals exposed to 700 ppm D4 were increased 18, 20, and 22% over controls while PB-treated animals showed increases of 33, 27, and 27% over controls on days 6, 13, and 27 respectively. Hepatic incorporation of BrdU following exposure to D4 was highest on day 6 (labeling index = 15-22%) and was at or below control values by day 27. This pattern of transient hyperplasia was observed in all hepatic lobes examined and was similar to the pattern observed following treatment with PB.

A physiologically based toxicokinetic model for lake trout (Salvelinus namaycush).

A physiologically based toxicokinetic (PB-TK) model for fish, incorporating chemical exchange at the gill and accumulation in five tissue compartments, was parameterized and evaluated for lake trout (Salvelinus namaycush). Individual-based model parameterization was used to examine the effect of natural variability in physiological, morphological, and physico-chemical parameters on model predictions. The PB-TK model was used to predict uptake of organic chemicals across the gill and accumulation in blood and tissues in lake trout. To evaluate the accuracy of the model, a total of 13 adult lake trout were exposed to waterborne 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE), concurrently, for periods of 6, 12, 24 or 48 h. The measured and predicted concentrations of TCE, PCE and HCE in expired water, dorsal aortic blood and tissues were generally within a factor of two, and in most instances much closer. Variability noted in model predictions, based on the individual-based model parameterization used in this study, reproduced variability observed in measured concentrations. The inference is made that parameters influencing variability in measured blood and tissue concentrations of xenobiotics are included and accurately represented in the model. This model contributes to a better understanding of the fundamental processes that regulate the uptake and disposition of xenobiotic chemicals in the lake trout. This information is crucial to developing a better understanding of the dynamic relationships between contaminant exposure and hazard to the lake trout.

Induction of hepatic xenobiotic metabolizing enzymes in female Fischer-344 rats following repeated inhalation exposure to decamethylcyclopentasiloxane (D5).

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane with a wide range of commercial applications. The present study was designed to investigate the effects of D5 on the expression and activity of selected rat hepatic phase I and phase II metabolizing enzymes. Female Fischer-344 rats were exposed to 160 ppm D5 vapors (6 h/day, 7 days/week, for 28 days) by whole-body inhalation. Changes in the activity and relative abundance of hepatic microsomal cytochromes P450 (CYP1A, CYP2B, CYP3A, and CYP4A), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were measured. Repeated inhalation exposure of rats to D5 increased liver size by 16% relative to controls by day 28. During a 14-day post-exposure period, liver size in D5-exposed animals showed significant recovery. Exposure to D5 did not change total hepatic P450, but increased the activity of hepatic NADPH-cytochrome c reductase by 1.4-fold. An evaluation of cytochrome P450 (CYP) enzymes in hepatic microsomes prepared from D5-exposed rats revealed a slight (1.8-fold) increase in 7-ethoxyresorufin O-deethylase (EROD) activity, but no change in immunoreactive CYP1A1/2 protein. A moderate increase (4.2-fold) in both 7-pentoxyresorufin O-depentylase (PROD) activity and immunoreactive CYP2B1/2 protein (3.3-fold) was observed. Testosterone 6beta-hydroxylase activity was also increased (2.4-fold) as was CYP3A1/2 immunoreactive protein. Although a small increase in 11- and 12-hydroxylation of lauric acid was detected, no change in immunoreactive CYP4A levels was measured. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D5-exposed group. UDPGT activity toward chloramphenicol was induced 1.8-fold, while no change in UDPGT activity toward 4-nitrophenol was seen. These results suggest that the profile for enzyme induction following inhalation exposure of female Fischer-344 rats to D5 vapors is similar to that reported for phenobarbital, and therefore D5 may be described as a weak "phenobarbital-like" inducer.

Evaluation of octamethylcyclotetrasiloxane (D4) as an inducer of rat hepatic microsomal cytochrome P450, UDP-glucuronosyltransferase, and epoxide hydrolase: a 28-day inhalation study.

Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

Vitamin E reduces oxidant injury to mitochondria and the hepatotoxicity of taurochenodeoxycholic acid in the rat.

BACKGROUND & AIMS: Hydrophobic bile acids have been implicated in the pathogenesis of cholestatic liver injury. The hypothesis that hydrophobic bile acid toxicity is mediated by oxidant stress in an in vivo rat model was tested in this study. METHODS: A dose-response study of bolus intravenous (i.v.) taurochenodeoxycholic acid (TCDC) in rats was conducted. Rats were then pretreated with parenteral alpha-tocopherol, and its effect on i.v. TCDC toxicity was evaluated by liver blood tests and by assessing mitochondrial lipid peroxidation. RESULTS: Four hours after an i.v. bolus of TCDC (10 mumol/100 g weight), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels peaked, hepatic mitochondria showed evidence of increased lipid peroxidation, and serum bile acid analysis was consistent with a cholestatic injury. Liver histology at 4 hours showed hepatocellular necrosis and swelling and mild portal tract inflammation. Treatment with parenteral alpha-tocopherol was associated with a 60%-70% reduction in AST and ALT levels, improved histology, and a 60% reduction in mitochondrial lipid peroxidation in rats receiving TCDC. CONCLUSIONS: These data show that hepatocyte injury and oxidant damage to mitochondria caused by i.v. TCDC can be significantly reduced by pretreatment with the antioxidant vitamin E. These in vivo findings support the role for oxidant stress in the pathogenesis of bile acid hepatic toxicity.

Dermal absorption of three waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus).

In vivo estimates of xenobiotic chemical flux across the dermal surface of intact fish were obtained by measuring chemical loss from venous blood to expired water. An experimental system was developed to separate the dermal route of exposure from all other routes. The system was then used to measure dermal absorption of tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE) in channel catfish (Ictalurus punctatus) and rainbow trout (Oncorhynchus mykiss), two fish with very different skin anatomies. The kinetics of accumulation varied among chemicals, but for each compound were similar among species. TCE accumulated rapidly, reaching steady state in blood within 48 hr. Steady state was not reached in 48 hr with PCE or HCE, although blood levels of PCE were probably close to steady-state values. Dermal flux estimates (based on branchial efflux) for TCE, PCE, and HCE were two to four times greater in catfish than in trout. Arterial blood concentrations of each compound were three to six times greater in catfish. These observations are indicative of greater flux across catfish skin, augmented by higher blood:water chemical partitioning. Trout skin is covered with scales and has no taste buds, while catfish skin does not possess scales and has numerous taste bud papillae. Both scales and taste bud papillae originate in the dermis and extend to the skin surface through the epidermis. In catfish these taste buds may offer channels through which chemicals diffuse across the epidermis to the more vascularized dermis. A comparison of dermal and branchial uptake was made by estimating zero-time dermal and branchial fluxes for all three chloroethanes. The mean dermal fluxes for TCE, PCE, and HCE ranged from 1.4 to 2.8, 1.8 to 3.6, and 1.4 to 3.2% of the total flux (branchial plus dermal) in rainbow trout and channel catfish, respectively. This research demonstrates that dermal absorption of waterborne chemicals occurs in large adult fish and results in distribution kinetics similar to those observed in inhalation exposures. Compared to branchial uptake, the dermal route of exposure appears to be relatively unimportant in large fish. It may, however, be very important in smaller fish and for juveniles of larger species.

A physiologically based toxicokinetic model for dermal absorption of organic chemicals by fish.

A physiologically based toxicokinetic model was developed to describe dermal absorption of waterborne organic chemicals by fish. The skin was modeled as a discrete compartment into which compounds diffuse as a function of chemical permeability and the concentration gradient. The model includes a countercurrent description of chemical flux at fish gills and was used to simulate dermal-only exposures, during which the gills act as a route of elimination. The model was evaluated by exposing adult rainbow trout and channel catfish to hexachloroethane (HCE), pentachloroethane (PCE), and 1,1,2,2-tetrachloroethane (TCE). Skin permeability coefficients were obtained by fitting model simulations to measured arterial blood data. Permeability coefficients increased with the number of chlorine substituent groups, but not in the manner expected from a directly proportional relationship between dermal permeability and skin:water chemical partitioning. An evaluation of rate limitations on dermal flux in both trout and catfish suggested that chemical absorption was limited more by diffusion across the skin than by blood flow to the skin. Modeling results from a hypothetical combined dermal and branchial exposure indicate that dermal uptake could contribute from 1.6% (TCE) to 3.5% (HCE) of initial uptake in trout. Dermal uptake rates in catfish are even higher than those in trout and could contribute from 7.1% (TCE) to 8.3% (PCE) of initial uptake in a combined exposure.

alpha-tocopherol ameliorates oxidant injury in isolated copper-overloaded rat hepatocytes.

The objective of this study was to determine the role of oxidant stress in cell injury produced by in vivo copper overload of isolated rat hepatocytes. Rats were maintained on diets with elevated or normal copper content, and hepatocytes were isolated and then incubated for 4 h in physiologic buffer at physiologic oxygen saturations. In hepatocytes from copper-overloaded rats, a significant loss of cell viability (trypan blue exclusion) over 4 h compared with control cells was associated with a significant increase in lipid peroxidation (thiobarbituric acid-reacting substances). Incubation of copper-overloaded hepatocytes with the copper chelator, 2,3,2-tetramine, had a partial protective effect. Incubation with D-alpha-tocopheryl succinate completely ameliorated the copper-induced changes in viability and lipid peroxidation. We conclude that antioxidants may protect the isolated hepatocyte from copper toxicity and should be explored as potential therapeutic agents in states of copper overload.

Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids.

BACKGROUND & AIMS: The mechanisms causing liver injury in cholestatic diseases are unclear. The hypothesis that accumulation of hydrophobic bile acids in hepatocytes during cholestasis leads to generation of oxygen free radicals and oxidative injury was tested. The aim of this study was to determine if hydrophobic bile acid toxicity is associated with increased hydroperoxide generation in isolated rat hepatocytes and mitochondria. METHODS: Hepatocytes were exposed to taurochenodeoxycholic acid (TCDC; 0-2000 mumol/L) or taurocholic acid (TC; 1000 mumol/L), and cellular injury, intracellular hydroperoxide generation, and thiobarbituric acid-reacting substances (TBARS) were measured. Isolated mitochondria were incubated with 400 mumol/L chenodeoxycholic acid or 400 mumol/L cholic acid, and hydroperoxide generation was measured fluorometrically. RESULTS: Hepatocyte injury, hydroperoxide generation, and TBARS increased over 4 hours on exposure to TCDC but not TC. Hydroperoxide generation preceded hepatocyte injury and accumulation of TBARS. Preincubation of hepatocytes with the antioxidant, d-alpha-tocopheryl succinate, completely abrogated cellular injury, hydroperoxide, and TBARS generation. Hydroperoxide generation was increased in mitochondria exposed to chenodeoxycholic acid. CONCLUSIONS: Intracellular generation of hydroperoxides by mitochondria appears to be an early event in hydrophobic bile acid-induced hepatocyte toxicity. Antioxidants may be of benefit in cholestasis.

Oxidant injury to hepatic mitochondria in patients with Wilson's disease and Bedlington terriers with copper toxicosis.

BACKGROUND/AIMS: Copper overload leads to liver injury in humans with Wilson's disease and in Bedlington terriers with copper toxicosis; however, the mechanisms of liver injury are poorly understood. This study was undertaken to determine if oxidant (free radical) damage to hepatic mitochondria is involved in naturally occurring copper toxicosis. METHODS: Fresh liver samples were obtained at the time of liver transplantation from 3 patients with Wilson's disease, 8 with cholestatic liver disease, and 5 with noncholestatic liver disease and from 8 control livers. Fresh liver was also obtained by open liver biopsy from 4 copper-overloaded and 4 normal Bedlington terriers and from 8 control dogs. Hepatic mitochondria and microsomes (humans only) were isolated, and lipid peroxidation was measured by lipid-conjugated dienes and thiobarbituric acid-reacting substances. In humans, liver alpha-tocopherol content was measured. RESULTS: Lipid peroxidation and copper content were significantly increased (P < 0.05) in mitochondria from patients with Wilson's disease and copper-overloaded Bedlington terriers. More modest increases in lipid peroxidation were present in microsomes from patients with Wilson's disease. Mitochondrial copper concentrations correlated strongly with the severity of mitochondrial lipid peroxidation. Hepatic alpha-tocopherol content was decreased significantly in Wilson's disease liver. CONCLUSIONS: These data suggest that the hepatic mitochondrion is an important target in hepatic copper toxicity and that oxidant damage to the liver may be involved in the pathogenesis of copper-induced injury.

In vitro and in vivo studies on the degradation of metallothionein.

Degradation of metallothionein (MT) from rat liver was examined. Degradation of apo-MT by liver homogenate was greater than that by cytosol. At pH 5.5, degradation by homogenate was more than that at pH 7.2. These findings suggest that proteases that function at acidic pH are probably involved in MT degradation. Because lysosomes are the principal subcellular organelles that contain acid proteases (cathepsins), we compared the degradation of apo-MT by lysosomes and cytosol. Apo-MT was degraded about 400 times faster by lysosomal fraction than by cytosolic fraction. To determine the relative importance of different cathepsins, we used different inhibitors. Leupeptin, which inhibits cathepsins B and L, inhibited the degradation of apo-MT by 80%, implying that cathepsins B and/or L might be very important in the intracellular turnover of MT. Cathepsin D appeared to be the least significant, because apo-MT degradation was reduced by about 20% by inhibiting cathepsin D. When we extended this study with purified cathepsins, we obtained the same answer, i.e., the ability of different cathepsins to degrade apo-MT was in the following order: cathepsin B >> cathepsin C > cathepsin D. While apo-MT was susceptible to degradation, ZnMT and CdMT were highly resistant to degradation. Coincubation of ZnMT or CdMT with either lysosomal extract or purified cathepsins did not result in any appreciable degradation even after 16 hr. However, longer incubations did result in some degradation, especially by purified cathepsin B. Interestingly, CdMT degraded little faster than ZnMT by both lysosomal extract as well as purified cathepsin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of metallothionein by superantigenic bacterial exotoxin: probable involvement of the immune system.

Metallothionein (MT) can be induced in mouse liver by a bacterial exotoxin, toxic shock syndrome toxin-1 (TSST-1). Hepatic MT was induced by TSST-1 in a dose-dependent manner from 100 micrograms/kg through 3 mg/kg in CF-1 mice, and by 6 h the induction was almost maximal. The increase of hepatic MT occurred at the mRNA level, also, and both MT-I and II mRNAs increased coordinately. Because TSST-1 is a superantigen, it was investigated whether TSST-1 induces MT through cytokines as a consequence of immunostimulation. In low-cytokine-producing mice (C3H/HeJ), up to a dose of 1 mg/kg of TSST-1, there was only 2- to 3-fold increase of hepatic MT. In contrast, in normal-cytokine-producing mice (C3Heb/FeJ), TSST-1 increased MT in a dose-dependent manner, and at a dose of 1 mg/kg, there was a 25-fold increase in hepatic MT. This suggests that activation of the immune system is probably involved in the induction of MT by TSST-1. Studies on the role of specific hepatic cytokines (IL-1, TNF-alpha, and IL-6) in TSST-mediated hepatic MT induction showed that TSST-1 did not increase hepatic IL-1 or TNF-alpha significantly over controls in any of the mouse strains studied. In contrast, TSST-1 induced hepatic IL-6 in all three strains of mice. However, in CF-1 and C3Heb/FeJ mice (normal-cytokine-producing) IL-6 induction preceded MT mRNA induction, but in C3H/HeJ mice (low-cytokine-producing), IL-6 induction did not precede MT mRNA induction.

Differential expression of the metallothionein gene in liver and brain of mice and rats.

Expression of the metallothionein I (MT-I) gene was studied in liver and brain of control mice and rats, as well as following administration of Cd and lipopolysaccharide (LPS). Time-course studies revealed that MT mRNA reached a maximum in liver of both mice and rats 6 hr following treatment with Cd or LPS. MT mRNA from control and Cd- and LPS-treated rat brains could not be detected by Northern-blot analysis of total RNA, but Northern analysis with poly(A)-enriched RNA revealed that induction of MT mRNA in rat brain does occur with both Cd and LPS treatment. In contrast, mouse brain MT mRNA was easily detected by Northern-blot analysis of total RNA. It was also clear from Northern-blot analyses of both mouse and rat brain that LPS induced more MT mRNA than did Cd. Quantitation of MT mRNA by solution hybridization revealed that Cd and LPS induced similar amounts of MT mRNA in livers of mice (about 0.64 fmol/micrograms total RNA by Cd and 0.68 by LPS) and rats (about 0.23 fmol/micrograms total RNA by Cd and 0.21 by LPS). Therefore, both inducers increased MT mRNA about threefold more in mouse liver than in rat liver. In mouse and rat brain, LPS induced about twice as much MT mRNA as did Cd (about 0.08 fmol/micrograms total RNA by Cd and 0.16 by LPS in mice and about 0.006 fmol/micrograms total RNA by Cd and 0.008 by LPS in rats). However, the actual amount of MT mRNA induced in rat brain by either inducer was minimal compared to that in mouse brain. In fact, Cd induced 13 times more MT mRNA in mouse brain than in rat brain, and LPS induced about 20 times more MT mRNA in mouse brain than in rat brain. Cd distribution to liver was similar in both mice and rats, but the Cd concentration in mouse brain was about 60% more than that in rat brain. Distribution of LPS was also similar in mouse and rat livers, as well as in mouse and rat brains. Therefore, there exists a difference in the expression of MT gene in both liver and brain of mice and rats, the expression in mice being higher than that in rats. These findings suggest that such differential expression of the MT gene cannot be entirely accounted for by the difference in the tissue distribution of inducers. Other tissue-specific and species-specific factors controlling MT gene expression appear to be involved.

Induction of metallothionein by arsenicals in mice.

Metallothionein (MT) is a sulfhydryl-rich, metal-binding protein that provides protection against metal toxicity. MT is induced by acute stress, hormones, metals, and various organic compounds. Recently, arsenicals have also been shown to induce MT. However, the mechanism and character of MT induction by arsenicals is unknown. Therefore, the effect of various arsenic forms on the tissue concentration of MT was determined. Mice were injected sc with various doses of arsenite [As(III)], arsenate [As(V)], monomethylarsenate (MMAA), and dimethylarsenate (DMAA), and MT content in the liver was measured 24 hr later by the Cd-hemoglobin radioassay. As(III) is a potent hepatic MT inducer in that a 30-fold increase in MT was observed at the dose of 85 mumol/kg. In comparison, it took 3-, 50-, and 120-fold higher molar amounts of As(V), MMAA, and DMAA, respectively to produce a similar effect. MMAA produces the largest increase in hepatic MT (80-fold), followed by As(III) (30-fold), As(V) (25-fold), and DMAA (10-fold). However, none of the arsenicals induced MT in mouse primary hepatocyte cultures. Both MT-I and MT-II were coordinately induced by As(III), As(V), and MMAA. MT induction by As(III) was further characterized following sc administration of arsenite (85 mumol/kg). Hepatic MT induction peaked at 24 hr, and in addition to the liver, As(III) also increased MT in kidney, spleen, stomach, intestine, heart, and lung. MT-I mRNA increased 24-, 52-, and 11-fold at 3, 6, and 15 hr after As(III) administration. This induction profile is similar to that observed after Zn or Cd exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo microdialysis sampling of phenol and phenyl glucuronide in the blood of unanesthetized rainbow trout: implications for toxicokinetic studies.

Microdialysis (MD) is a sampling method that allows continuous in vivo collection of free, unbound chemicals in blood and interstitial fluids. In the present study we describe a surgical method for placement of a MD probe in the dorsal aorta of 600- to 900-g rainbow trout (Onchorhynchus mykiss). A specially designed probe guide was inserted into the dorsal aorta via the mouth. A PE-50 polyethylene cannula was then inserted into the probe guide and used to further position and maintain the probe guide in the dorsal aorta. Once proper placement of the probe guide was ascertained, the cannula was removed and a CMA-10 MD probe (4-mm tip) was inserted. The animal was then placed into a respirometer-metabolism chamber and allowed to recover from anesthesia. The placement and functionality of the probe were evaluated by examining the in vivo toxicokinetics of phenol (PH) and phenyl glucuronide (PG) in the blood of an unanesthetized rainbow trout exposed to water-borne PH (7.0 mg/liter). Prior to and following the introduction of PH into the metabolism chamber, MD samples (150 microliters) were collected at 30-min intervals and analyzed for free plasma PH and PG by HPLC. Total PH in exposure water and blood was also monitored every 30 min. Free PH and total PH in plasma accumulated rapidly and reached apparent steady-state levels of 54 and 142 pmol/microliters, respectively, in about 60 min. A blood:water partition coefficient of 2.0-2.6 was determined from these data, while bound and free plasma PH were 60 and 40%, respectively. PG was not detected until approximately 90 min of PH exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of hepatic metallothionein by paraquat.

Paraquat, a frequently used contact herbicide, produces oxidative stress by undergoing redox cycling and generating reactive oxygen species. Paraquat is also effective at increasing hepatic levels of metallothionein (MT). The mechanism(s) by which agents that induce oxidative stress produce increases in MT concentrations is not yet known. Therefore, the goal of the current study was to characterize the elevation in hepatic MT produced by paraquat administration to mice and to examine potential mechanism(s) of this increase. A dose-response study for increases in MT showed that administration of 0.1 to 0.5 mmol/kg of paraquat, sc, increased hepatic MT with a maximal increase of 36-fold. Subsequent studies were carried out with paraquat at a dose (0.3 mmol/kg, sc) that caused oxidative stress, as shown by a 35-fold increase in the biliary excretion of oxidized glutathione. There were coordinate elevations of both hepatic MT-I and MT-II mRNA of approximately 5-fold with peaks at both 6 and 24 hr after paraquat. The time course for the elevation in hepatic MT protein following paraquat treatment showed that MT levels had a maximal increase of 18-fold obtained at 36 hr. Paraquat appears to be an indirect MT inducer, in that there were no elevations in MT when cultured mouse hepatocytes were exposed to paraquat. No rise in liver Zn was observed prior to the increase in hepatic MT, thus, a Zn redistribution to the liver did not cause the increase in hepatic MT following paraquat administration. Adrenalectomy did not abolish the increase in MT produced by paraquat, suggesting that adrenal gland products are not required for the increase in MT produced by paraquat. In conclusion, the chemical mediator responsible for the increase in hepatic MT after paraquat was not determined, but the elevation in MT concentration appears to be due to increased transcription.

In vitro degradation of apo-, zinc-, and cadmium-metallothionein by cathepsins B, C, and D.

Metallothionein (MT) has been extensively studied over the past several years because of its probable role in endogenous metal homeostasis and cellular protection. A large body of knowledge now exists describing the physicochemical properties of MT as well as the mechanisms involved in MT induction. It has been well established that MT protects tissues from metal toxicity by chelating metals that would otherwise be available to interact with and disrupt vital cell functions. Information on the degradation of metal-saturated MT and the fate of the metals associated with it would be extremely important in predicting metal toxicity. Lysosomes have been targeted as a possible subcellular site for the turnover of MT; however, the susceptibility of MT to degradation by specific acidic proteases (i.e., cathepsins) has not been described. Therefore, the purpose of the present study was to examine the relative abilities of cathepsins B, C, and D to degrade Zn7-MT, Cd7-MT, and apo-MT in vitro. In so doing, the effects of metal species, degree of metal saturation, and pH on the degradation processes were evaluated. Time course experiments revealed that apo-MT was rapidly degraded by all three cathepsins. Cathepsin B degraded apo-MT approximately 36-fold more rapidly than cathepsin C and 45-fold more rapidly than cathepsin D. Therefore, under the in vitro conditions used in this study, the relative potency of the cathepsins tested was cathepsin B much much greater than cathepsin C greater than cathepsin D. In comparison, metal-saturated MT was more than 1000-fold more resistant to degradation by the cathepsins tested. In order to determine how much metal was needed to protect MT against degradation, apo-MT was reconstituted with increasing molar equivalents of Zn2+. The results suggest that as metal to apo-MT ratios increase, less apo-MT substrate is available to the protease and degradation decreases.

Role of hepatic lysosomes in the degradation of metallothionein.

The degradation of metallothionein (MT) by rat liver was examined. Degradation of MT by liver homogenate was greater than by cytosol. In addition, MT degradation by the homogenate at pH 5.5 was more than that at pH 7.2. Because lysosomal proteases function at acidic pH, these findings suggest the importance of lysosomes in MT degradation. The degradation by the lysosomal fraction was about 400-fold greater than that by the cytosol. Because cathepsins are the principal lysosomal proteases, we used cathepsin-specific inhibitors, such as leupeptin, E-64 and pepstatin, to determine the relative importance of different cathepsins in degrading MT. The study reveals that cathepsin B and/or L is (are) probably the most important enzyme(s) in degrading hepatic MT, because leupeptin, which blocks cathepsin B and L activity, inhibited the degradation of apo-MT by about 80%. Cathepsin D appears to be of least importance in MT degradation, because inhibition of this enzyme by pepstatin reduced degradation by only 20%. Studies on the degradation of apo-MT, ZnMT, and CdMT indicated that apo-MT is about 1500-fold more sensitive to degradation than ZnMT and CdMT. These data suggest that metals protect MT from degradation. This is further supported by a reconstitution experiment, which shows that with a progressive decrease of MT: metal ratio following titration of apo-MT by metals, there is a concomitant reduction in degradation. At a lysosomal pH of around 4.7, about 60% of Zn and 20% of Cd are displaced from MT, thereby making it susceptible to degradation. We propose, therefore, that lysosomes are probably important for MT degradation in vivo and that metal release is a prerequisite for degradation. With the release of metals, MT becomes susceptible to degradation, which is probably accomplished by the lysosomal cathepsins, in particular cathepsins B and L.