D. Stuart Wyand

Ultrastructural Changes during Acute Acetaminophen-Induced Hepatotoxicity in the Mouse: A Time and Dose Study

This study was undertaken to evaluate the early ultrastructural changes during the development of acetaminophen hepatotoxicity. Doses at or near the threshold for hepatotoxicity were selected to permit comparison of early reversible effects to those which ultimately progressed to necrosis in the absence of early agonal effects or drug-induced mortality. Both 300-and 600-mg/kg doses resulted in similar declines in hepatic glutathione levels to 14 and 22% of control values, respectively, by 2 hours, with more rapid recovery after the low dose. Plasma sorbitol dehydrogenase activity was elevated after 600 mg/kg but not after 300 mg/kg. During the first 2 hours after acetaminophen there was cytomegaly with rapid progression to necrosis after 600 mg/kg but minimal progression after 300 mg/kg. Ultrastructurally, vesiculation, vacuolation and mitochondrial and plasma membrane degeneration culminated in scattered single cell death by 4 hours and widespread centrilobular necrosis by 8 hours after 600 mg/kg. The time course of lesion development was slower after 300 mg/kg with damage restricted to the first two to three rows of centrilobular cells and limited numbers of isolated necrotic cells by 8 hours. By 18 to 24 hours livers of mice given 300 mg/kg appeared normal. Results are consistent with the endoplasmic reticulum being the site of acetaminophen activation and initial attack. However, early ultrastructural changes in mitochondria and plasma membrane observed after the high dose were not prominent after the low dose. This suggests that early acetaminophen damage to these organelles may play a critical role in acetaminophen hepatotoxicity.

Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity

Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithelium. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5-microns paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

Extrahepatic Lesions Induced by Acetaminophen in the Mouse

Acetaminophen in acute overdose is primarily recognized as potentially hepatotoxic with few descriptions of extrahepatic lesions other than nephrotoxicity. Fasted adult, male mice, both standard and germ-free, were given acetaminophen orally and killed at selected times, from 30 minutes to 48 hours after treatment. In addition to the expected hepatic effects after 600 mg acetaminophen/kg, degenerative and necrotic changes were found in four non-hepatic tissues. Nephrosis developed 2 to 4 hours after treatment and paralleled the course of hepatic damage. Necrosis of bronchiolar epithelium in the absence of inflammation was evident 4 to 6 hours after acetaminophen administration as was onset of testicular changes. Spermatidic degeneration with early development of spermatidic multinucleated giant cells were characteristic features. Areas of lymphoid necrosis were also visible in splenic follicles and Peyers patches 18 to 24 hours after treatment. These observations have demonstrated that other tissues in addition to liver and kidney are damaged by acetaminophen toxicity and should be considered in cases of acetaminophen overdosage.

Selective protein arylation and the age dependency of acetaminophen hepatotoxicity in mice

Male CD-1 mice 1, 1.5, 2, and 3 months old were given 600 mg of acetaminophen (APAP)/kg, po, and liver damage was assessed 12 hr later. The most severe hepatotoxicity was in 3-month-old mice, while the other age groups exhibited little damage. The onset of susceptibility to APAP hepatotoxicity did not correlate with the level of activity of the mixed-function oxidase system as assessed in vitro, since drug metabolizing capability was similar between 2- and 3-month-old mice. Through 4 hr after administration of APAP to 2- and 3-month-old mice in vivo, glutathione (GSH) depletion and both plasma and liver APAP concentrations were similar between ages. Additionally, 24 hr after dosing, 3-month-old mice excreted marginally more APAP-glucuronide conjugate and parent compound in urine than 2-month-old animals, while both age groups excreted similar amounts of the APAP-sulfate and GSH-derived conjugates. Even though the extent of binding of radioactive APAP to macromolecules at 4 hr was similar between 2- and 3-month-old animals, the pattern of immunochemically targetted cytosolic and microsomal proteins was different. Thus, in APAP exposure the extent of binding to specific proteins rather than the overall amount of covalent binding may be the critical determinant of the hepatotoxic response. In the present study, the age-related differences in susceptibility to APAP-induced hepatotoxicity were related to the differences in selective protein arylation.

Evidence against deacetylation and for cytochrome P450-mediated activation in acetaminophen-induced nephrotoxicity in the CD-1 mouse

Acetaminophen (APAP) administration (600 mg/kg, po) results in proximal tubular necrosis in 18-hr fasted, 3-month-old male CD-1 mice. This study was undertaken to determine if deacetylation of APAP to p-aminophenol (PAP) is a prerequisite to nephrotoxicity in the mouse, as it is in the Fischer rat. Administration of either APAP or PAP to mice resulted in significant elevations of plasma urea nitrogen and marked proximal tubular necrosis at 12 hr after dosing. Prior inhibition of APAP deacetylation by the carboxylesterase inhibitors bis(p-nitrophenyl) phosphate or tri-o-tolyl-phosphate did not alter APAP hepatotoxicity or nephrotoxicity. By contrast, pretreatment with the MFO inhibitor piperonyl butoxide decreased APAP nephrotoxicity but not that of PAP. Immunochemical analysis of kidneys from APAP-treated mice demonstrated covalently bound APAP but no binding was detected after mice were treated with a nephrotoxic dose of PAP. Since the antibody used has been characterized as being directed primarily against the N-acetyl moiety of bound APAP metabolite and since it did not react with kidney proteins of mice given a nephrotoxic dose of PAP, it is unlikely that APAP deacetylation preceded binding or that acetylation of bound PAP occurred. Taken together, these findings indicate that in the CD-1 mouse, APAP-induced nephrotoxicity differs from that previously described for the Fischer rat and likely involves cytochrome P450-dependent activation and subsequent covalent binding of a metabolite without prior deacetylation.

Protection against acetaminophen-induced hepatotoxicity by prior treatment with fenitrothion

Abstract This study was undertaken to determine if interactions of toxicologic importance might occur during simultaneous exposure of mice to acetaminophen and fenitrothion. Acetaminophen administration induced dose-dependent hepatic morphologic changes which were accompanied by leakage of sorbitol dehydrogenase (SDH) into plasma 18 hr after dosing. Glutathione (GSH) depletion was maximal 2 hr after acetaminophen even at doses below those required for hepatic necrosis. Pretreatments with fenitrothion (50 or 500 mg/kg, ip) decreased hepatic GSH concentration and inhibited microsomal aniline hydroxylase and paranitroanisole O -demethylase activities. Similar treatments in mice subsequently challenged with acetaminophen (400 or 600 mg/kg, po) prevented the elevations in plasma SDH activity and lethality. Massive necrosis and 70% lethality were observed after 800 mg acetaminophen/kg and these effects were prevented by earlier pretreatment with 50 mg fenitrothion/kg. This pretreatment also decreased the degree of hepatic GSH depletion detected at 2 hr after 800 mg of acetaminophen. This finding would suggest that production of acetaminophen electrophile had been deceased by the pretreatment. Thus, with respect to interaction with acetaminophen, these results suggest that fenitrothion inhibition of mixed function oxidases predominated over fenitrothion depletion of GSH. Thus, even though GSH stores were lowered, less acetaminophen electrophile was generated and critical macromolecules were spared.

In Vivo and in Vitro Evidence for in Situ Activation and Selective Covalent Binding of Acetaminophen (APAP) in Mouse Kidney

Acetaminophen (APAP, N-acetyl-p-aminophenol) is a widely used analgesic and antipyretic which, at high doses, causes acute hepatic centrilobular necrosis in man and a variety of laboratory animals (Proudfoot and Wright, 1970; Boyer and Rouff, 1971; Mitchell et al., 1973). In addition, acute renal proximal tubular necrosis following APAP has been reported in man (Kleinman et al., 1980; Cobden et al., 1982; Kher and Makker, 1987; Davenport and Finn, 1988). A similar lesion has been described in the Fischer rat (McMurtry et al., 1978; Newton et al., 1983) andthe CD-1 mouse (Placke et al, 1987) but the APAP metabolite responsible for the toxicity is different between species. In the rat, APAP is enzymatically deacetylated to paminophenol, a potent nephrotoxicant whose activation is independent of cytochrome P450 (Crowe et al., 1979; Calder et al., 1979; Newton et al., 1982; 1985a; 1985b). By contrast, enzymatic deacetylation of APAP is not required in the mouse but instead activation of intact APAP by cytochrome P450 appears to mediate nephrotoxicity (Bartolone et al, 1989; Emeigh Hart et al., 1989a; 1989b). Since hepatic metabolism of APAP is similarly dependent on cytochrome P450 (Mitchell et al, 1973), nephrotoxicity and renal adduct accumulation could arise from transport of a livergenerated metabolite or adduct to the kidney and not from in situ metabolism of APAP. The purpose of the present study was to determine, using renal proximal tubule (RPT) cell suspensions and immunohistochemistry, if the mouse kidney could generate such adducts in situ.

Spontaneous Glioblastoma Multiforme in a Charles River CD Rat

The purpose of this report is to describe the occurrence and pathology of a spontaneous and rare brain tumor in a strain of rat commonly employed for drug and chemical safety testing. Glioblastoma multiforme is another term for “malignant astrocytoma” (Grade 3 or 4) and is characterized by cellular and nuclear hyperchromasia and pleomorphism, multinucleated giant cells and pallisading necrosis. The tumor exhibits highly aggressive invasive behavior. In contrast, “anaplastic glioma” is a term applied to any malignant tumor of glial cells including those containing astrocytes. Often, there are variable mixtures of cell types within a given tumor. None of these anaplastic gliomas exhibit the degree of anaplasia or invasive behavior seen in the tumor of this report. Primary brain tumors in rats are infrequent compared to tumors found in other organ systems. In a recent report, astrocytoma was the most frequent glial cell tumor in Sprague-Dawley rats with an incidence of 28/2,630 in males and 18/2,765 in females (3). This literature report included a total of 88 brain tumors from 5,395 untreated controls and 297 brain tumors from a total of 19,251 rats examined in the combined control and treated groups. None of the astrocytomas reported satisfied the criteria needed for designation as glioblastoma multiforme. A 1974 report of 38 naturally occurring brain tumors found in 4 1,000 Sprague-Dawley rats over a 5-year period included 10 astrocytomas or

The effect of ethanol consumption on trace mineral status in elderly rats

Abstract The effect of ethanol consumption on trace mineral status in 23-month old female rats was investigated. Ethanol was isocalorically substituted for dextrin at 35% of the calories in a liquid diet. Tissue trace minerals were determined by atomic absorption spectrophotometry. Compared to controls, rats consuming ethanol for 30 days had decreased liver concentrations of zinc, iron and magnesium. Other tissues examined were unaffected. Tissue concentrations of copper and manganese and hemoglobin, hematocrit and plasma ceruloplasmin were not different. Histological liver examination revealed fatty infiltration in ethanol treated rats. Decrease of liver concentrations of zinc, iron and magnesium in the absence of cirrhosis is attributed to the effect of ethanol and was not due to lower dietary intake of these minerals in the ethanol consuming animals compared to the controls.