Differential induction of heme oxygenase-1 in macrophages and hepatocytes during acetaminophen-induced hepatotoxicity in the rat: effects of hemin and biliverdin

Heme oxygenase-1 (HO-1), also known as heat shock protein 32, has been shown to protect against oxidant-induced tissue injury. In the present studies, we analyzed expression of this enzyme in macrophages and hepatocytes following acetaminophen administration and its potential role in hepatotoxicity. Treatment of rats with a hepatotoxic dose of acetaminophen (1 g/kg, ip) resulted in a time-dependent induction of HO-1 in the liver. This was observed within 6 h of acetaminophen administration in both hepatocytes and macrophages. Hepatocytes were found to be more sensitive than macrophages to the effects of acetaminophen on HO-1. Up regulation of HO-1 in the liver following acetaminophen administration correlated with induction of ferritin and manganese superoxide dismutase (MnSOD). To determine if HO-1 was hepatoprotective, rats were pretreated with hemin (30 micromol/kg, ip), a potent inducer of the enzyme. Following hemin treatment, we observed a time-dependent increase in HO-1 protein in the liver and in serum bilirubin levels. Pretreatment of rats with hemin was found to prevent acetaminophen-induced hepatotoxicity, as measured histologically and biochemically by decreased serum transaminase levels. This was correlated with more rapid increases in expression of hepatic ferritin and MnSOD. Heme metabolism via HO-1 generates biliverdin, which is rapidly converted to bilirubin by biliverdin reductase. Pretreatment of rats with biliverdin (40 micromol/kg, ip) was also found to block acetaminophen-induced injury. These data suggest that HO-1 is an important component of antioxidant defense during acetaminophen-induced hepatotoxicity.     

The effect of imipramine after single and repeated administration on the apomorphine response in the acoustic startle reflex in rats.

The paper presents the results of studies into the effects of single and repeated (2 or 10 mg/kg po, twice daily for 14 days) administration of imipramine (IMI) on the behavior of male Wistar rats in an acoustic startle response test. Statistically significant attenuation of the animal reactivity was observed after 8 or 14 days administration of IMI in a dose of 10 mg/kg. Apomorphine APO (1.0 mg/kg, ip) enhanced the animals reactivity after repeated (but not single) administration of IMI in a dose of 2 mg/kg. The obtained results confirm the hypothesis about an increase of the dopaminergic system reactivity under the influence of repeated administration of tricyclic antidepressants.     

Changes in hepatic glutathione concentration modify cadmium-induced hepatotoxicity

Cd has a strong affinity for sulfhydryl groups and is hepatotoxic. Thus, to further understand the mechanism of Cd-induced liver injury, the effect of increased and decreased hepatic glutathione (GSH) concentration on Cd-induced liver injury was examined. Liver GSH was lowered by pretreating rats with phorone (250 mg/kg, ip) or diethyl maleate (0.85 mg/kg, ip) 2 hr prior to challenge with various doses of Cd. Ten hours after Cd (1) 40–80% of the rats pretreated with phorone or diethyl maleate and challenged with 1.0–2.0 mgCd/kg died whereas no mortality was observed in the control group; (2) plasma enzyme activities of alanine (ALT) and aspartate (AST) aminotransferase and sorbitol dehydrogenase (SDH) were markedly increased in phorone and diethyl maleate-pretreated rats challenged with Cd (0.7–2.0 mg/kg) versus control rats; and (3) moderate changes in liver histology were observed in corn oil pretreated and Cd challenged rats, while prior depletion of GSH potentiated histopathologic changes in liver produced by Cd alone. Another group of rats received cysteine (1.9 g/kg, po) 3 hr prior to injection of a lethal dose of Cd. Cysteine pretreatment increased liver GSH levels by 22% 3 hr after administration and attenuated Cd-induced liver injury as evidenced by marked decreases in plasma ALT, AST, and SDH activities. Pathological changes in liver were also reduced. These data indicate that liver reduced GSH concentration is important in modulating Cd-induced hepatotoxicity.     

Effects of route of administration and repetitive dosing on the disposition kinetics of di(2-ethylhexyl) phthalate and its mono-de-esterified metabolite in rats

The disposition kinetics of the plasticizer di(2-ethylhexyl) phthalate (DEHP) and its biologically active metabolite mono(2-ethylhexyl) phthalate (MEHP) were studied in rats following single or multiple administration of DEHP by various routes. Following a single intraarterial (ia) injection, a large apparent volume of distribution (5390 ml/kg) and a high rate of clearance (21.5 ml/min/kg) were observed for DEHP. The systemic availability of DEHP was low following both single po (13.6%) and ip (5.2%) administration. A marked route-dependency in the formation of MEHP from DEHP was observed. The circulating concentrations of MEHP were substantially higher than those of DEHP (i.e., area under the blood concentration-time curve (AUC) ratio of approximately 7) after po administration, whereas concentrations of the mono-de-esterified metabolite were much lower relative to the parent diester concentration after ia or ip administration (i.e., AUC ratio less than 0.4). Pharmacokinetic calculations revealed that approximately 80% of a po dose of DEHP undergoes mono-de-esterification, as compared to only about 1% of the dose following either ia or ip administration. Hence, the low po systemic availability of DEHP may be largely attributed to presystemic hydrolysis of DEHP to MEHP in the gut, whereas slow and/or incomplete absorption is the likely cause of the poor bioavailability of DEHP after ip administration. No significant accumulation in the circulating concentrations of DEHP or derived MEHP were observed following 7 days of repetitive administration of DEHP. However, multiple ip injections resulted in an apparent decrease in the rate and/or extent of DEHP absorption from the peritoneal cavity, while no significant change in the po absorption of the diester was observed. The striking difference in the MEHP to DEHP AUC ratio between po and ip routes was still evident after multiple dosing. These data suggest that previously reported differences in the biologic effects of DEHP in rodents following different routes of administration may be due to route dependency in the mono-de-esterification of the diester.     

Distribution, elimination, and test for carcinogenicity of 2,6-dinitrotoluene after intraperitoneal and oral administration to strain a mice

After intraperitoneal (ip) and oral (po) administration of 2,6-dinitrotoluene (2,6-DNT), the distribution, elimination, and lung tumor response were determined in strain A mice. In a 30-wk bioassay and at total ip doses of 600, 1500, or 3000 mg/kg, or total po doses of 1200, 3000, or 6000 mg/kg, 2,6-DNT did not produce an increase in lung adenomas when compared to vehicle-treated controls. The urine was the major route of elimination of both ip and po administered 3H-labeled 2,6-DNT, with 87.6, 55.2, and 49.1% of ip doses of 1, 10, and 100 mg/kg, respectively, excreted within 4 h. The corresponding amounts excreted after po administration were 33.6, 25.2, and 24.3%, which increased to 53.7, 53.5, and 48.6% after 8 h. The distribution of 2,6-DNT in various tissues (blood, liver, kidneys, lungs, small and large intestine) showed no evidence for preferential uptake or retention at any of the ip or po doses. Terminal half-lives of radioactive material in the liver were 1.11, 0.95, and 1.16 h after ip doses of 1, 10, and 100 mg/kg, respectively. At all ip doses (1, 10, and 100 mg/kg), rapid and extensive metabolism of 3H-labeled 2,6-DNT was observed, as judged by the low amounts (less than 2% of the total 3H/tissue) of unchanged 3H-labeled 2,6-DNT that could be reisolated from blood, liver, small intestine, or lungs at 2 h after administration. The extent of 3H-labeled 2,6-DNT metabolism by these tissues after po administration was similar, except for the relatively high amounts of unchanged 3H-labeled 2,6-DNT (approximately 50% of the total 3H/tissue between 1 and 8 h after 100 mg/kg) present in the small intestine. At these times, negligible (less than 2%) unchanged 3H-labeled 2,6-DNT was present in the large intestine. It is concluded that ip or po administered 2,6-DNT is not carcinogenic in the strain A mouse lung-tumor bioassay. Excretion in the urine is the major mode of elimination after either ip or po administration, but is slower after po than after ip dosing. It is likely that the liver and small intestine are major sites of 2,6-DNT metabolism, and it is possible that po administered 2,6-DNT is also metabolized by the microflora of the large intestine.     

Effects of iron overload on bile secretion and hepatic porphyrin metabolism in ethinyl estradiol-treated rats

We investigated the effects of ethinyl estradiol (5 mg/kg body wt daily for 5 days, orally) and/or iron sorbitol (50 mg/kg body wt daily for 5 days, i.m.) on bile flow, bile salt independent fraction (BSIF), hepatic delta-aminolevulinate synthase (ALA-S) and uroporphyrinogen decarboxylase (URO-D) in female rats. Ethinyl estradiol administration was associated with a significant decrease of bile flow and BSIF and an increase in URO-D activity in comparison to control values. Iron alone did not modify biliary parameters, but significantly increased the activity of ALA-S. Combined treatment with ethinyl estradiol plus iron partially corrected the reduction of BSIF and restored the activity of ALA-S and URO-D to control levels. Thus iron appears to exert a partially protective effect against ethinyl estradiol-induced cholestasis. No porphyrinogenic effect was observed.     

The possible role of the ethanol-inducible isozyme of cytochrome P450 in the metabolism and distribution of carbon disulfide

Acute treatment with ethanol and other alcohols has been shown to potentiate the hepatotoxicity of certain xenobiotics, in part via induction of the mixed-function oxidase (MFO) system. Carbon disulfide (CS2)-induced hepatotoxicity and inhibition of the MFO system have been shown to be a consequence of MFO metabolism. In the present study, the ability of several different alcohols to induce the hepatic MFO metabolism of CS2 and the effects of this induction on CS2 distribution and hepatotoxicity were examined in rats. Eighteen hours after alcohol administration (1/2 LD50 dose, po), CS2 microsomal MFO metabolism was significantly enhanced, in order of descending potency, by isopropanol, methanol, and ethanol pretreatments, but not by isobutanol pretreatment. The degree of enhancement of CS2 metabolism by different alcohols paralleled the enhancement of nitroanisole O-demethylation and aniline hydroxylation, MFO activities associated with the ethanol-inducible isozyme of cytochrome P450. CS2 (1 mg/kg, ip, 3 hr) inhibited only the cytochrome P450-mediated activities enhanced by alcohol pretreatment. These results suggest that CS2 metabolism is catalyzed by the ethanol-inducible isozyme. Alcohol-induced rats had significantly more 14CS2-derived radioactivity in the liver than control and isobutanol-pretreated rats 3 hr after dosing (1 mg/kg, ip). However, only methanol pretreatment resulted in an increased retention of 14CS2-derived radioactivity in plasma, brain, and kidney. Unlike other alcohol pretreatments, methanol decreased the total 14C expired during the 3-hr period after CS2 dosing and caused a significant (twofold) increase in plasma glutamic-pyruvic transaminase, measured 24 hr after CS2 exposure (625 mg/kg). These data indicate that alcohol induction of MFO-dependent CS2 metabolism per se is not sufficient to result in CS2-induced hepatic damage although it does lead to loss of specific cytochrome P450 function.     

Tissue distribution of 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) and its effect on enzymes involved in tyrosine catabolism in the mouse

Administration of a single oral dose of 2-(2-nitro-4-trifluoromethyl-benzoyl)-cyclohexane-1,3-dione (NTBC) to mice increases the concentration of tyrosine in the plasma and aqueous humour. The tyrosinaemia is both time and dose-dependent with a single dose of 30 micromol NTBC/kg (10 mg/kg) producing maximal concentrations of tyrosine in plasma of about 1200 nmol/ml and in aqueous humour of about 2200 nmol/ml at 16 h after dosing. Analysis of the key hepatic enzymes involved in tyrosine catabolism, following a single dose of 30 micromol NTBC/kg, showed that 4-hydroxyphenylpyruvate dioxygenase (HPPD) was markedly inhibited soon after dosing and that the activity recovered very slowly. In response to the tyrosinaemia, the activity of hepatic tyrosine aminotransferase (TAT) was induced about two-fold, while the activity of hepatic homogentisic acid oxidase (HGO) was reduced at 4 and 5 days after dosing. Daily oral administration of NTBC at doses up to 480 micromol NTBC/kg (160mg/kg/day) to mice produced a maximal tyrosinaemia of about 600-700nmol/ml plasma, showing some adaptation relative to a single dose. Unlike the rat, no treatment-related corneal lesions of the eye were seen at any dose levels up to 6 weeks. Administration of a single oral dose of [14C]-NTBC at 30 micromol/kg led to selective retention of radiolabel in the liver and to a lesser extent the kidneys. Our studies show that NTBC is a potent inhibitor of mouse liver HPPD, which following repeat exposure produces a marked and persistent tyrosinaemia, which does not result in ocular toxicity.     

血红素氧合酶—1介导单磷酰酯A诱导的心脏延迟保护

目的:研究血红素氧合酶-Ⅰ(HO-Ⅰ)是否参与单磷酰酯A诱导的心脏延迟保护作用.方法:在体大鼠心脏缺血-再灌损伤模型,缺血前24小时应用单磷酰酯A(500μg/kg,ip)诱导心脏延迟保护,观察单磷酰酯A对心肌梗死面积、肌酸激酶(CK)活性以及血清一氧化氮(NO)浓度的影响,并检测心脏HO-ⅠmRNA和蛋白的表达.结果:单磷酰酯A明显缩小心梗面积,减少CK释放以及显著升高血清NO浓度(P<0.01).这些作用可被预先给予一氧化氮合酶抑制剂L-亚硝基精氨酸甲酯(L-NAME,10mg/kg,ip)和血红素氧合酶抑制剂锌原叶啉LX(ZnPP-9,45μmol/kg,ip)所取消(P<0.01);单磷酰酯A明显增加心脏HO-ⅠmRNA和蛋白的表达,这一作用不被L-NAME所影响(P>0.05).结论:HO-Ⅰ/NO途径介导单磷酰酯A诱导的延迟心脏保护.     

Curcumin attenuates dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase-1.

Curcumin (diferuloymethane), a yellow colouring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), has been reported to possess anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic activities. Curcumin exerts its chemoprotective and chemopreventive effects via multiple mechanisms. It has been reported to induce expression of the antioxidant enzymes in various cell lines. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a pivotal role in cytoprotection against noxious stimuli of both endogenous and exogenous origin. In the present study, we found that oral administration of curcumin at 200mg/kg dose for four consecutive days not only protected against dimethylnitrosamine (DMN)-induced hepatic injury, but also resulted in more than three-fold induction of HO-1 protein expression as well as activity in rat liver. Inhibition of HO-1 activity by zinc protoporphyrin-IX abrogated the hepatoprotective effect of curcumin against DMN toxicity. NF-E2-related factor 2 (Nrf2) plays a role in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase-2 detoxifying and antioxidant enzymes including HO-1. Curcumin administration resulted in enhanced nuclear translocation and ARE-binding of Nrf2. Taken together, these findings suggest that curcumin protects against DMN-induced hepatotoxicity, at least in part, through ARE-driven induction of HO-1 expression.     

Sulforaphane protects against acetaminophen-induced hepatotoxicity

Oxidative stress is closely associated with acetaminophen (APAP)-induced toxicity. Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, has been shown to protect against oxidant-induced tissue injury. This study investigated whether sulforaphane (SFN), as a HO-1 inducer, plays a protective role against APAP hepatotoxicity in vitro and in vivo. Pretreatment of primary hepatocyte with SFN induced nuclear factor E2-factor related factor (Nrf2) target gene expression, especially HO-1 mRNA and protein expression, and suppressed APAP-induced glutathione (GSH) depletion and lipid peroxidation, which eventually leads to hepatocyte cell death. A comparable effect was observed in mice treated with APAP. Mice were treated with 300 mg/kg APAP 30 min after SFN (5 mg/kg) administration and were then sacrificed after 6 h. APAP alone caused severe liver injuries as characterized by increased plasma AST and ALT levels, GSH depletion, apoptosis, and 4-hydroxynonenal (4-HNE) formations. This APAP-induced liver damage was significantly attenuated by pretreatment with SFN. Furthermore, while hepatic reactive oxygen species (ROS) levels were increased by APAP exposure, pretreatment with SFN completely blocked ROS formation. These results suggest that SFN plays a protective role against APAP-mediated hepatotoxicity through antioxidant effects mediated by HO-1 induction. SFN has preventive action in oxidative stress-mediated liver injury.     

Cystamine modulation of galactosamine-induced hepatotoxicity

The ability of cystamine treatment to protect against galactosamine-induced hepatic necrosis was investigated. Reduced hepatotoxicity was observed following galactosamine hydrochloride (400 mg/kg, ip) in male Sprague-Dawley rats that received cystamine dihydrochloride (300 mg/kg, po) 30 min prior to or 2, 4, 6, 8, or 12 hr after galactosamine. In contrast, uridine treatment only protected against galactosamine-induced liver damage if administered within 2 hr of galactosamine. Protection by cystamine was found to extend over 3 days during which time the lesion was resolving. The degree of protection was dose related when administered 12 hr after galactosamine. Cystamine did not prevent or alter the increase in hepatic Ca2+ seen following galactosamine administration. The results indicate that the protective effects of cystamine on galactosamine-induced hepatotoxicity are unrelated to prevention of the early biochemical events that initiate the injury. Furthermore, prevention of Ca2+ accumulation in damaged hepatocytes is not the mechanism of protection and hence is not necessarily an irreversible cytotoxic event.     

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in mice following acute oral administration of arsenate.

The relationship of exposure dose and tissue concentration of parent chemical and metabolites is a critical issue in cases where toxicity may be mediated by a metabolite or by parent chemical and metabolite acting together. This has emerged as an issue for inorganic arsenic (iAs), because both its trivalent and pentavalent methylated metabolites have unique toxicities; the methylated trivalent metabolites also exhibit greater potency than trivalent inorganic arsenic (arsenite, As(III)) for some endpoints. In this study, the time-course tissue distributions for iAs and its methylated metabolites were determined in blood, liver, lung, and kidney of female B6C3F1 mice given a single oral dose of 0, 10, or 100 micromol As/kg (sodium arsenate, As(V)). Compared to other organs, blood concentrations of iAs, mono- (MMA), and dimethylated arsenic (DMA) were uniformly lower across both dose levels and time points. Liver and kidney concentrations of iAs were similar at both dose levels and peaked at 1 h post dosing. Inorganic As was the predominant arsenical in liver and kidney up to 1 and 2 h post dosing, with 10 and 100 micromol As/kg, respectively. At later times, DMA was the predominant metabolite in liver and kidney. By 1 h post dosing, concentrations of MMA in kidney were 3- to 4-fold higher compared to other tissues. Peak concentrations of DMA in kidney were achieved at 2 h post dosing for both dose levels. Notably, DMA was the predominant metabolite in lung at all time points following dosing with 10 micromol As/kg. DMA concentration in lung equaled or exceeded that of other tissues from 4 h post dosing onward for both dose levels. These data demonstrate distinct organ-specific differences in the distribution and methylation of iAs and its methylated metabolites after exposure to As(V) that should be considered when investigating mechanisms of arsenic-induced toxicity and carcinogenicity.     

Impact of repeated exposure on toxicity of perchloroethylene in Swiss Webster mice

The aim was to study the subchronic toxicity of perchloroethylene (Perc) by measuring injury and repair in liver and kidney in relation to disposition of Perc and its major metabolites. Male SW mice (25-29g) were given three dose levels of Perc (150, 500, and 1000 mg/kg day) via aqueous gavage for 30 days. Tissue injury was measured during the dosing regimen (0, 1, 7, 14, and 30 days) and over a time course of 24-96h after the last dose (30 days). Perc produced significant liver injury (ALT) after single day exposure to all three doses. Liver injury was mild to moderate and regressed following repeated exposure for 30 days. Subchronic Perc exposure induced neither kidney injury nor dysfunction during the entire time course as evidenced by normal renal histology and BUN. TCA was the major metabolite detected in blood, liver, and kidney. Traces of DCA were also detected in blood at initial time points after single day exposure. With single day exposure, metabolism of Perc to TCA was saturated with all three doses. AUC/dose ratio for TCA was significantly decreased with a concomitant increase in AUC/dose of Perc levels in liver and kidney after 30 days as compared to 1 day exposures, indicating inhibition of metabolism upon repeated exposure to Perc. Hepatic CYP2E1 expression and activity were unchanged indicating that CYP2E1 is not the critical enzyme inhibited. Hepatic CYP4A expression, measured as a marker of peroxisome proliferation was increased transiently only on day 7 with the high dose, but was unchanged at later time points. Liver tissue repair peaked at 7 days, with all three doses and was sustained after medium and high dose exposure for 14 days. These data indicate that subchronic Perc exposure via aqueous gavage does not induce nephrotoxicity and sustained hepatotoxicity suggesting adaptive hepatic repair mechanisms. Enzymes other than CYP2E1, involved in the metabolism of Perc may play a critical role in the metabolism of Perc upon subchronic exposure in SW mice. Liver injury decreased during repeated exposure due to inhibition of metabolism and possibly due to adaptive tissue repair mechanisms.     

Modulation by heme and zinc protoporphyrin of colonic heme oxygenase-1 and experimental inflammatory bowel disease in the rat

Reactive oxygen species, suggested to be involved in inflammatory bowel disease, may be modulated by endogenous anti-oxidant products of heme oxygenase-1 (HO-1). In the present work, HO-1 expression in trinitrobenzene sulphonic acid (TNBS)-induced colitis in the rat and the effects of HO-1 modulation, particularly by the HO-1 inducer, heme, were further evaluated. Colitis was induced by intracolonic challenge with TNBS and assessed macroscopically and by myeloperoxidase (MPO) assay. Heme oxygenase activity was determined by measurement of bilirubin formation and HO-1 protein expression was determined by Western blotting. TNBS challenge led to an early and substantial induction of HO-1 protein expression and heme oxygenase activity in the colon that peaked after 48-72 h and declined over 10 days. Heme (30 micromol/kg/day, s.c) increased colonic HO-1 protein expression and enzyme activity and decreased colonic damage and myeloperoxidase activity. Short-term administration of cadmium chloride (2 mg/kg, s.c.), another known HO-1 inducer, also reduced the colonic injury and myeloperoxidase levels. In contrast, the HO-1 inhibitor, zinc protoporphyrin (50 micromol/kg/day, s.c) significantly increased the colonic damage and myeloperoxidase activity over 10 days, as did tin protoporphyrin (30 micromol/kg/day, s.c). These results support the proposal that induction of HO-1 provides a protective mechanism in this model under both acute and more-chronic conditions, and that its selective up-regulation could thus be of therapeutic potential in colitis.     

Enhanced hepatotoxicity and toxic outcome of thioacetamide in streptozotocin-induced diabetic rats

Diabetes is known to potentiate thioacetamide (TA)-induced liver injury via enhanced bioactivation. Little attention has been given to the role of compensatory tissue repair on ultimate outcome of hepatic injury in diabetes. The objective of this study was to investigate the effect of diabetes on TA-induced liver injury and lethality and to investigate the underlying mechanisms. We hypothesized that hepatotoxicity of TA in diabetic rats would increase due to enhanced bioactivation-mediated liver injury and also due to compromised compensatory tissue repair, consequently making a nonlethal dose of TA lethal. On day 0, male Sprague-Dawley rats (250-300 g) were injected with streptozotocin (STZ, 60 mg/kg ip) to induce diabetes. On day 10 the STZ-induced diabetic rats and the nondiabetic rats received a single dose of TA (300 mg/kg ip). This normally nonlethal dose of TA caused 90% mortality in the STZ-induced diabetic rats. At various times (0-60 h) after TA administration, liver injury was assessed by plasma alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), and liver histopathology. Liver function was evaluated by plasma bilirubin. Cell proliferation and tissue repair were evaluated by [(3)H]thymidine ((3)H-T) incorporation and proliferating cell nuclear antigen (PCNA) assays. In the nondiabetic rat, liver necrosis peaked at 24 h and declined thereafter toward normal by 60 h. In the STZ-induced diabetic rat, however, liver necrosis was significantly increased from 12 h onward and progressed, culminating in liver failure and death. Liver tissue repair studies showed that, in the liver of nondiabetic rats, S-phase DNA synthesis was increased at 36 h and peaked at 48 h following TA administration. However, DNA synthesis was approximately 50% inhibited in the liver of diabetic rats. PCNA study showed a corresponding decrease of cell-cycle progression, indicating that the compensatory tissue repair was sluggish in the diabetic rats. Further investigation of tissue repair by employing equitoxic doses (300 mg TA/kg in the non-diabetic rats; 30 mg TA/kg in the diabetic rats) revealed that, despite equal injury up to 24 h following injection, the tissue repair response in the diabetic rats was much delayed. The compromised tissue repair prolonged liver injury in the diabetic rats. These studies suggest that the increased TA hepatotoxicity in the diabetic rat is due to combined effects of increased bioactivation-mediated liver injury of TA and compromised compensatory tissue repair.     

Time course of cadmium-induced ultrastructural changes in rat liver

Ultrastructural changes in rat liver were studied 1, 2, 4, 6, 8, and 10 hr after administration of a single, high dose of Cd (3.9 mg Cd/kg, iv) or after repeated administration of a lower dose (0.5 mg Cd/kg, sc, 6 days/week for 6 months). These dosing regimens have been previously shown to produce hepatotoxicity and result in large accumulations of Cd in liver. In addition to light and electron microscopy, plasma enzyme activities indicative of liver injury, namely alanine (ALT) and aspartate (AST) aminotransferase, were determined at the aforementioned times. One hour after an acute dose of Cd, electron photomicrographs of liver showed dilation of the rough endoplasmic reticulum with concomitant loss of membrane-associated ribosomes, nucleolar condensation, and an increase in the number of perichromatin granules. At later times (4 and 6 hr), ultrastructural changes included mitochondrial swelling associated with matrical inclusions, further dilation and vesiculation of rough endoplasmic reticulum, and presence of a fibrillar material within cytoplasm. In contrast to changes observed after single administration of Cd, the predominant hepatic lesions in rats injected repeatedly with the metal over 6 months were interstitial fibrosis, nuclear enlargement, and an increase in number and predominance of nucleoli. Ultrastructural evidence of nuclear alterations included condensation of nucleoli and an increase in the number of perichromatin granules. These results indicate that Cd interferes with hepatic protein synthesis early after injection of a large dose, and that further degenerative changes occur later and possibly in response to protein inhibition. Although severe degenerative changes in liver were not evident in rats chronically exposed to the metal, Cd-induced changes in nuclei and nucleoli also indicate the likelihood of altered protein synthesis.     

Prior administration of a low dose of thioacetamide protects type 1 diabetic rats from subsequent administration of lethal dose of thioacetamide

Previously, we reported that an ordinarily non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic rats due to inhibited liver tissue repair, whereas 30 mg TA/kg allows 100% survival due to stimulated although delayed tissue repair. Objective of this investigation was to test whether prior administration of a low dose of TA (30 mg/kg) would lead to sustainable stimulation of liver tissue repair in type 1 diabetic rats sufficient to protect from a subsequently administered lethal dose of TA. Therefore, in the present study, the hypothesis that preplacement of tissue repair by a low dose of TA (30 mg TA/kg, ip) can reverse the hepatotoxicant sensitivity (autoprotection) in type 1 diabetic rats was tested. Preliminary studies revealed that a single intraperitoneal (ip) administration of TA causes 90% mortality in diabetic rats with as low as 75 mg/kg. To establish an autoprotection model in diabetic condition, diabetic rats were treated with 30 mg TA/kg (priming dose). Administration of priming dose stimulated tissue repair that peaked at 72h, at which time these rats were treated with a single ip dose of 75 mg TA/kg. Our results show that tissue repair stimulated by the priming dose enabled diabetic rats to overexpress, calpastatin, endogenous inhibitor of calpain, to inhibit calpain-mediated progression of liver injury induced by the subsequent administration of lethal dose, resulting in 100% survival. Further investigation revealed that protection observed in these rats is not due to decreased bioactivation. These studies underscore the importance of stimulation of tissue repair in the final outcome of liver injury (survival/death) after hepatotoxicant challenge. Furthermore, these results also suggest that it is possible to stimulate tissue repair in diabetics to overcome the enhanced sensitivity of hepatotoxicants.     

THE TEMPORAL RELATIONSHIP BETWEEN BACTERIAL LIPOPOLYSACCHARIDE AND MONOCROTALINE EXPOSURES INFLUENCES TOXICITY: SHIFT IN RESPONSE FROM HEPATOTOXICITY TO NITRIC OXIDE-DEPENDENT LETHALITY

Liver injury from a variety of hepatotoxicants, including the food-borne phytotoxin monocrotaline (MCT), can be augmented by exposure to a noninjurious dose of the inflammagen bacterial lipopolysaccharide (LPS). In a previous study, a nontoxic dose of LPS given 4 h after MCT resulted in synergistic hepatotoxicity within 12-18 h. This study was designed to determine whether temporal differences in MCT and LPS exposure affect toxicity. When LPS (3.4 2 10 6 EU/kg; iv) was given one hour before MCT (100 mg/kg; ip), hepatotoxicity developed between 4 and 8 h after MCT administration, and mortality was much greater than when LPS was administered 4 h after MCT. To explore this difference, the temporal relationship between LPS and MCT exposure (7.4 2 10 6 EU/kg and 100 mg/kg, respectively) was altered. Twenty-four-hour survival was high in animals that received LPS 4 h before (86%) or after (88%) MCT, but it decreased markedly when LPS was administered 1 h before MCT (17%). Using this latter dosing regimen, animals became moribund as early as 4 h after MCT administration. Since liver injury was similar from regimens that differed greatly in mortality, death appeared to result from extrahepatic causes. To explore a role for nitric oxide (NO)-induced shock in this regimen, animals were treated with aminoguanidine (AG), an inhibitor of inducible NO synthase, prior to administration of LPS given an hour before MCT. In the cotreated animals, AG significantly attenuated mortality and decreased plasma nitrate/nitrite concentrations, markers of NO biosynthesis. Hence, the primary target of toxicity from MCT and LPS cotreatment appeared to shift from the liver to an extrahepatic site or sites as exposure to these agents occurred closer together temporally. NO appears to be causally involved in the deaths of animals treated with LPS 1 h before MCT.     

Comparative studies of huperzine A, donepezil, and rivastigmine on brain acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine levels in freely-moving rats

AIM: To assess the effects of cholinesterase inhibitors huperzine A, donepezil and rivastigmine on cerebral neurotransmitters in the cortex and hippocampus in freely-moving rats. METHODS: Double-probe cerebral microdialysis and HPLC with electrochemical detection were used to detect neurotransmitters. RESULTS: Our results showed that huperzine A (0.25, 0.5, and 0.75 micromol/kg, po) dose-dependently elevated extracellular acetylcholine (ACh) levels in the medial prefrontal cortex (mPFC) and hippocampus. Oral administration of donepezil (5.4 micromol/kg) or rivastigmine (1 micromol/kg) also elicited significant increases in ACh in the mPFC and hippocampus. The time course of cortical acetylcholinesterase (AChE) inhibition with the 3 inhibitors mirrored the increases of ACh at the same dose. The marked elevation of ACh after oral administration of huperzine A (0.5 micromol/kg) and donepezil (5.4 micromol/kg) was associated with a significantly increased release of dopamine (DA) in the mPFC or hippocampus. None of the 3 inhibitors affected norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels in the mPFC and hippocampus. The effects of huperzine A and rivastigmine did not depend on the route of administration, but donepezil was less efficacious by the oral route than by ip injection. The ability of huperzine A to increase ACh levels was unchanged when tests were performed after multiple oral administration of the drug at 0.5 micromol/kg, once per day for 30 d. CONCLUSION: The present findings showed that, in molar terms, huperzine A had similar potency on increasing mPFC ACh and DA levels as compared to the 11- and 2-fold dosages of donepezil and rivastigmine, respectively, and had longer lasting effects after oral dosing.