Endotoxin pretreatment protects against the hepatotoxicity of acetaminophen and carbon tetrachloride: role of cytochrome P450 suppression.

Bacterial endotoxin (lipopolysaccharide, LPS) is known to potentiate the toxicity of many hepatotoxicants. However, exposure to a sublethal dose of LPS renders animals tolerant to a lethal dose of LPS, and protects against the toxicity of some chemicals. This study was designed to examine the effects of LPS pretreatment on acetaminophen- and carbon tetrachloride (CCl(4))-induced liver injury in LPS-sensitive C3H/OuJ and LPS-resistant C3H/HeJ mice. Pretreatment of male C3H/OuJ mice with a single injection of LPS (0. 1 mg/kg, ip, for 24 h) protected against the hepatotoxic effects of acetaminophen (400 mg/kg) and carbon tetrachloride (CCl(4), 30 mg/kg), as indicated by serum alanine aminotransferase activity. In contrast, pretreatment of C3H/HeJ mice with 0.1 or 10 mg/kg LPS afforded no protection against the hepatotoxic effects of acetaminophen and CCl(4). In an attempt to determine the mechanism of LPS-induced protection against acetaminophen- and CCl(4)-induced hepatotoxicity in C3H/OuJ mice, liver cytochrome P450 was determined 24 h after LPS injection. LPS treatment caused a 26% decrease in total P450 content in C3H/OuJ but not in C3H/HeJ mice. CYP3A-catalized testosterone 6 beta-, 2 beta-, and 15 beta-hydroxylation was decreased 40% by LPS only in C3H/OuJ mice. To determine whether the differences to LPS-response in the two stains of mice is mediated by a strain-related difference in the release of cytokines, mice were pretreated with interleukin-1 (IL-1 alpha, 5 x 10(5) U/mouse), and the hepatoprotection and hepatic P450 enzymes were examined. IL-1 alpha pretreatment equally protected against the hepatotoxicity of acetaminophen and CCl(4) in both strains, and suppressed the total microsomal P450 and P450 enzyme-catalyzed testosterone hydroxylation to a similar extent. In conclusion, LPS pretreatment suppressed hepatic cytochrome P450 enzymes and protected against the hepatotoxicity of acetaminophen and CCl(4) in LPS-sensitive C3H/OuJ mice, but not in LPS-refractory C3H/HeJ mice. This protective effect of LPS appears to be mediated through the release of cytokines such as IL-1 alpha, which in turn suppresses the cytochrome P450 responsible for the activation of acetaminophen and CCl(4) to reactive metabolites.

Pharmacokinetic, pharmacodynamic, and pharmacotoxic profiles of recombinant-methionyl human interleukin-2[alanine-125] (r-metHuIL-2[ala-125]) following intravenous and subcutaneous administration in rats.

Comparative pharmacotoxicity studies in rats were performed to evaluate the response to r-metHuIL-2[ala-125] following 2 or 4 weeks of daily intravenous or subcutaneous administration, as well as to evaluate pharmacokinetic and pharmacodynamic responses. Pharmacokinetic analysis indicated that r-metHuIL-2[ala-125] showed high bioavailability and nonlinear concentration profiles. Pharmacodynamic responses to intravenous or subcutaneous dosing with r-metHuIL-2[ala-125], as measured by white blood cell counts, were comparable. Preclinical safety studies (6, 30, and 150 micrograms kg-1 day-1) indicated that r-metHuIL-2[ala-125], whether given intravenously or subcutaneously, was associated with increased circulating and infiltrating levels of lymphocytes and eosinophils. Bone marrow lymphoid hyperplasia and splenic extramedullary hematopoiesis were similarly observed in each study. This pattern of effects was considered an exaggerated pharmacodynamic response to r-metHuIL-2[ala-125]. Of further note was a histopathologic finding described as hepatocyte single cell necrosis which was observed following both intravenous and subcutaneous administration and was considered to be a toxic response to high doses of r-metHuIL-2[ala-125]. The no observable adverse effect level (NOAEL) for r-metHuIL-2[ala-125] via intravenous administration was 6 micrograms kg-1 day-1, while that for subcutaneous administration was 30 micrograms kg-1 day-1. Data herein present a form of rHuIL-2 with pharmacokinetic and pharmacodynamic profiles that are similar when given by these two systemic routes. Pharmacotoxic data, based on NOAELs, suggest that subcutaneous administration may be a preferred clinical route of administration.

Cd-metallothionein nephrotoxicity in inbred strains of mice.

Genetic differences in the acute hepatic and testicular toxicity of Cd occur among different strains of mice. However, it is not known whether genetic variation to the renal damage caused by Cd-metallothionein (CdMT) exists. Therefore, male mice of the C3H/HeJ, C57/Bl10, CBA/CA, and DBA/2J strains, previously shown to differ in hepatic and testicular injury due to Cd, were treated with CdMT at dosages of 0.2, 0.4, 0.8, and 1.6 mg/kg (sc). For all strains of mice, tissue accumulation of Cd occurred predominantly in kidney, which had two to three times as much Cd as liver, while testes had no measurable amounts of Cd. Hepatic and renal metallothionein (MT) concentrations were increased with increasing dosage of CdMT, and no differences between strains were demonstrated. Urinary glucose was increased significantly at the three highest dosages of CdMT, with no differences between strains. At each dose level, light microscopic manifestations of CdMT nephropathy did not differ between strains. In summary, all CdMT-treated strains of mice responded similarly with respect to all measured renal parameters (accumulation of Cd and MT and nephrotoxicity). Unlike the strain differences in hepatic and testicular injury from Cd in these strains of mice, CdMT nephrotoxicity shows no such genetic variation.

Endotoxin induction of hepatic metallothionein is mediated through cytokines.

Metallothionein (MT), a low molecular-weight, cysteine-rich, metal-binding protein, is induced by many environmental factors and a variety of stimuli. Bacterial endotoxin (lipopolysaccharide, LPS) injection is experimentally used to produce acute stress and is an effective inducer of hepatic MT. However, the mechanism of LPS induction of MT is not known. In the present studies, we used two substrains of mice, differing in their production of cytokines after LPS administration, to test the hypothesis that MT induction by LPS is mediated through cytokines. Normal (C3Heb/FeJ) and low cytokine-producing (C3H/HeJ) mice were given various doses of LPS, interleukin-1 (IL-1), interleukin-6 (IL-6), or tumor necrosis factor (TNF), and hepatic MT was determined 24 hr later by the Cd/hemoglobin assay. The low-cytokine-producing mice were much less responsive to the induction of MT by LPS (50 vs 150 micrograms MT/g liver after 1.0 mg LPS/kg, ip) than the normal mice, but were equally responsive to the induction of MT by IL-1 (0.03-1.0 microgram/mouse). IL-6 (0.5-5.0 micrograms/mouse), and TNF (0.005-0.5 microgram/mouse). All the cytokines produced a dose-dependent increase of hepatic MT levels in these two murine substrains (up to five- to sevenfold over controls). In conclusion, these data suggest that LPS induction of MT may be mediated through cytokines.

Effect of sulfhydryl-deficient diets on hepatic metallothionein, glutathione, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) levels in rats.

Low dietary concentrations of methionine and cysteine are known to decrease hepatic glutathione content. However, it is not known if restricting the dietary content of these sulfur containing amino acids also affects hepatic levels of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the cofactor for sulfation, or metallothionein, a protein rich in sulfhydryl groups. Rats were fed diets lacking cysteine and containing various concentrations of methionine (0.15, 0.3, or 0.6%) for 8 days. Control diet contained 0.3% each of methionine and cysteine. Hepatic glutathione levels were decreased approximately 75% in rats fed diets containing 0.15 or 0.3% methionine. In contrast, PAPS and hepatic metallothionein concentrations were not decreased by the low sulfhydryl diets. Additionally, rats on the various diets were challenged by the administration of ZnCl2 (3 mmol/kg. sc). In both control rats and rats maintained on sulfhydryl-deficient diets, ZnCl2 increased hepatic metallothionein to the same level. However, significantly lower levels of PAPS were observed after ZnCl2 in rats receiving sulfhydryl-deficient diets than in controls. In summary, restriction of dietary sulfhydryl markedly decreases the hepatic content of glutathione and has a minor effect on PAPS concentration, but does not decrease the basal hepatic concentration of metallothionein or its induction by ZnCl2.

A review of the toxicity and carcinogenicity of anthraquinone derivatives.

Anthraquinones (AQs) are a group of functionally diverse chemicals structurally related to anthracene. Both natural and synthetic AQs have widespread applications throughout industry and medicine, thereby indirectly and directly exposing the human population. Because of the close similarity in structure between AQs and the toxic analogue, anthracene, there is concern over the potential damage which these compounds may produce. This review summarizes the toxicity and carcinogenicity of synthetically-derived AQs in experimental animals, classified into 3 categories based upon phenolic, amino, or nitro substitution to the AQ ring structure. The effect of chemical substitution in relation to toxicity and carcinogenicity is discussed.

Comparisons of the toxicity of CdCl2 and Cd-metallothionein in isolated rat hepatocytes.

In the intact animal, inorganic Cd distributes mainly to the liver and produces hepatotoxicity, while Cd-metallothionein (CdMT) distributes primarily to the kidney and produces nephrotoxicity. CdMT has also been demonstrated to be more toxic than Cd in cultured kidney cells, but it is not known if CdMT is more toxic to all cultured cells or if there is a good correlation between in vitro and in vivo toxicity. Therefore, hepatocytes, which were isolated and grown in monolayer culture for 24 h, were incubated with CdCl2 (1-100 microM) or CdMT (3-100 microM Cd). The intracellular K+ content was quantitated 24 h later as an index of toxicity. The K+ concentration of the hepatocytes was decreased 50% by 4 microM CdCl2, whereas 25 microM CdMT was required to produce similar injury. In the intact animal, zinc induces the synthesis of MT and decreases the hepatotoxicity of Cd. ZnCl2 added to the media (100 microM) for 24 h before exposure to Cd or CdMT increased the intracellular MT concentration 700%. This elevation in MT reduced the toxicity of CdCl2 approximately 80% but did not alter the toxicity of CdMT. In summary, CdCl2 is more toxic to cultured hepatocytes than Cd-MT, and MT induction decreases the toxicity of CdCl2 in hepatocytes, as has been observed in the intact animal. This indicates that cultured hepatocytes appear to be an excellent model for examining the hepatotoxicity of Cd.

Progressive lung injury over a one-year period after a single inhalation exposure to beryllium sulfate.

The chronic pulmonary toxicity of beryllium sulfate was examined in rats over a 1-yr period after a single, 1-h exposure. Male rats, exposed in a nose-only inhalation chamber to an aerosol of 4.05 micrograms Be/L, were evaluated for lung toxicity by the methods of bronchoalveolar lavage, lung cell kinetics, and histopathologic analysis. Bronchoalveolar lavage activities for alkaline phosphatase (Alk Pase) and acid phosphatase (Ac Pase) were elevated 3 wk after exposure; lactate dehydrogenase (LDH) and Alk Pase activities peaked 3 months after exposure. Histopathologic analysis revealed progressive focal interstitial pneumonitis with a prominent alveolar component of heteromorphic macrophages, neutrophils, and debris. No increase was noted in the overall labeling index in the alveolar cell population at any of the time points sampled. This study demonstrates the effectiveness of bronchoalveolar lavage fluid analysis in monitoring lung damage over a prolonged period and shows that the pulmonary toxicity of beryllium manifests itself as a progressive lesion from a single 1-h inhalation exposure to BeSO4.

Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmium-metallothionein.

Acute exposure to Cd produces liver injury, whereas chronic exposure results in kidney injury. Tolerance to the hepatotoxicity is observed during chronic exposure to Cd due to the induction of metallothionein (MT). The nephrotoxicity produced by chronic Cd exposure purportedly results from renal uptake of Cd-metallothionein (CdMT) synthesized in liver. The change in target organ from liver to kidney might be due to a lower amount of MT synthesized in the kidney in response to CdMT. Therefore, the purpose of the present study was to quantitate hepatic and renal MT induced by CdCl2 and CdMT. MT levels in mice were quantitated using the Cd-heme assay 24 hr after administration of CdCl2 (0.5-3.0 mg Cd/kg) and CdMT (0.1-0.5 mg Cd/kg). In both liver and kidney, MT reached higher levels following administration of CdCl2 (220 and 60 micrograms/g, respectively) than of CdMT (25 and 35 micrograms/g, respectively), probably because higher dosages of CdCl2 than CdMT are tolerated. CdMT produced 19 and 3 micrograms MT/micrograms Cd in liver and kidney, respectively, while CdCl2 produced 11 and 6 micrograms MT/micrograms Cd, respectively. In conclusion, induction of MT occurs in both the liver and kidney after administration of CdCl2 and CdMT. However, the kidney is less responsive than the liver to the induction of MT by both forms of Cd, which may contribute to making the kidney the target organ of toxicity during chronic Cd exposure.

Bronchoalveolar lavage in rats and mice following beryllium sulfate inhalation.

The effects of lung injury in rats and mice exposed to an aerosol of beryllium sulfate for 1 hr through nose-only inhalation were evaluated by the method of bronchoalveolar lavage. The lavage fluid of rats exposed to an aerosol of either 3.3 or 7.0 micrograms Be/liter over a 21-day period following exposure indicated lactate dehydrogenase (LDH) and alkaline phosphatase (Alk Pase) activities to be the most sensitive indicators of lung damage. LDH activity peaked at 8 days postexposure while Alk Pase activity was maximum at Day 5. Both values were 30 times greater than comparable controls at these time points. Acid phosphatase activity and albumin levels also increased over the 21-day period, but not to the same extent. The lung lavage of mice exposed to 7.2 micrograms Be/liter showed LDH activity as the most sensitive indicator of lung damage with a maximum response three times greater than that of controls at Day 5.

Protection by parenteral iron administration against the inhalation toxicity of beryllium sulfate.

Animals exposed to an aerosol of BeSO4 showed a significant reduction in mortality with iron treatment. Rats were exposed for 2 h in a nose-only inhalation chamber for 14 days to an aerosol of 2.59 micrograms/Be/l. The cumulative mortality of animals concurrently treated with iron salt was significantly reduced (P less than 0.05) compared to animals which had not received iron treatment.

Acute pulmonary toxicity of beryllium sulfate inhalation in rats and mice: cell kinetics and histopathology.

The acute response in the lung of mice and rats exposed to an aerosol of BeSO4 was characterized by cell kinetics and histopathology. Animals were exposed for 1 hour in a nose-only chamber to a 13 microgram/liter chamber concentration of BeSO4 or to H2SO4 as control aerosol, and killed thereafter over a period of 21 days. Lung cell kinetics were evaluated as the labeling index (LI), defined as the percentage of cells labeled with tritiated thymidine. The labeled cells were also differentiated as to type. In rats, the LI showed a peak response on Day 8 following exposure, while the LI of mice showed a maximum response at Day 5. In rats, the proliferative response involved type II alveolar epithelial cells, interstitial and capillary endothelial cells. Histopathology showed type II alveolar cell hyperplasia and vacuolization of their cytoplasm, a thickened interstitium with infiltrates of interstitial macrophages and segmental leukocytes; a prominent feature was alveolar macrophages with ragged membranes. Three weeks after exposure, the interstitial response was largely resolved. In mice, the proliferative response was mainly found in the alveolar macrophage population and the interstitial and endothelial cells. Histopathological changes were similar to those found in rats, although less severe.

Ferritin and in vivo beryllium toxicity.

Beryllium (Be+2), a divalent metal ion, is toxic to both man and animal. Although the molecular basis for its toxicity is unknown, it is well established that micromolar concentrations of beryllium specifically inhibit certain enzymes. Previous in vitro studies have shown that the presence of ferritin, an iron-storage protein, reactivated these enzymes by sequestering beryllium (Price and Joshi, 1984). In the present study we demonstrate in vivo that beryllium and zinc are bound by ferritin in greater amounts than Pb+2, Cu+2, and Cd+2. Beryllium did not induce the synthesis of metallothionein. In animals pretreated with an iron salt (ferric ammonium citrate, 40 mg/kg body wt), liver ferritin was elevated approximately five times and the toxicity of intravenously injected beryllium was significantly attenuated. Excretion and deposition studies suggested that iron salt treatment resulted in a reduction of liver beryllium. Thus the protection against beryllium toxicity by ferric ammonium citrate may be due to increased production of ferritin which binds beryllium and its subsequent elimination in the feces.

The effect of cyclosporin A on pulmonary fibrosis induced by butylated hydroxytoluene, bleomycin and beryllium sulfate.

Interstitial pulmonary fibrosis is characterized by an abnormal accumulation of fibroblasts with a resultant increase in lung collagen content. Previous research has implied a possible involvement of the T-lymphocyte in this process. We used cyclosporin A (Cy A), a known immunosuppressant, to deplete T-lymphocyte-dependent responses in animals following treatment with agents known to produce fibrosis; butylated hydroxytoluene (BHT), bleomycin and beryllium (Be). BHT-treated mice and bleomycin-treated rats showed significant reduction in total lung hydroxyproline content with Cy A (P less than 0.05). These results suggest a contribution of the T-lymphocyte in the overall process of fibrosis, but do not indicate its role as the sole causative agent.