Genetic influences on the end-stage effector phase of arthritis.

K/BxN T cell receptor transgenic mice are a model of inflammatory arthritis, most similar to rheumatoid arthritis, that is critically dependent on both T and B lymphocytes. Transfer of serum, or just immunoglobulins, from arthritic K/BxN animals into healthy recipients provokes arthritis efficiently, rapidly, and with high penetrance. We have explored the genetic heterogeneity in the response to serum transfer, thereby focussing on the end-stage effector phase of arthritis, leap-frogging the initiating events. Inbred mouse strains showed clear variability in their responses. A few were entirely refractory to disease induction, and those which did develop disease exhibited a range of severities. F1 analyses suggested that in most cases susceptibility was controlled in a polygenic additive fashion. One responder/nonresponder pair (C57Bl/6 x NOD) was studied in detail via a genome scan of F2 mice; supplementary information was provided by the examination of knock-out and congenic strains. Two genomic regions that are major, additive determinants of the rapidity and severity of K/BxN serum-transferred arthritis were highlighted. Concerning the first region, on proximal chromosome (chr)2, candidate assignment to the complement gene C5 was confirmed by both strain segregation analysis and functional data. Concerning the second, on distal chr1, coinciding with the Sle1 locus implicated in susceptibility to lupus-like autoimmune disease, a contribution by the fcgr2 candidate gene was excluded. Two other regions, on chr12 and chr18 may also contribute to susceptibility to serum-transferred arthritis, albeit to a more limited degree. The contributions of these loci are additive, but gene dosage effects at the C5 locus are such that it largely dominates. The clarity of these results argues that our focus on the terminal effector phase of arthritis in the K/BxN model will bear fruit.

PML nuclear bodies are general targets for inflammation and cell proliferation.

Acute promyelocytic leukemia is associated with a t(15;17) translocation that generates a fusion product between PML and the retinoic acid receptor alpha. Recently, PML was shown to concentrate within subnuclear domains, referred to as nuclear bodies, that are disorganized in acute promyelocytic leukemia cells. This observation provided the first evidence that alteration of a nuclear structure may play a role in human pathogenesis. In an attempt to clarify the role of PML and, more generally, of the associated nuclear bodies, we used immunohistochemistry to explore the expression of PML in normal, inflammatory, and neoplastic human tissues. With the exception of endothelial cells and macrophages that contain a high amount of PML protein, a weak speckled labeling pattern was observed in the nucleus of all cell types analyzed. By contrast to normal tissues, the level of PML expression was considerably enhanced in inflammatory tissues, predominantly around the mononuclear cell infiltrate, as well as during either normal or pathological proliferative states, in particular in tumoral pathology. Surprisingly, in most hepatocellular carcinoma, a cytoplasmic delocalization of PML was observed. Finally, the number of PML nuclear bodies increased up to twice their normal value as quiescent cultured cells were stimulated to grow upon serum addition. Altogether these results strongly suggest that the PML-associated nuclear bodies are implicated both in the inflammatory process and in cell growth control.

Close correlation between beta-catenin gene alterations and nuclear accumulation of the protein in human hepatocellular carcinomas.

Several lines of evidence indicate that beta-catenin acquires oncogenic activity when its intracellular concentration increases as a result of either mutation in the beta-catenin gene itself or inactivation of the adenomatous polyposis coli (APC) gene. In an attempt to elucidate the molecular mechanisms underlying hepatocellular carcinogenesis, we have studied the frequency of beta-catenin gene alterations in exon 3, a region known to represent a mutation hot spot, and its inappropriate protein expression by immunohistochemistry in 73 hepatocellular carcinomas (HCCs). The results were correlated with different clinical and pathological data, particularly with the presence or not of an associated cirrhosis. Fourteen (19%) HCCs showed beta-catenin gene alterations with missense mutations in nine cases and interstitial deletions in five cases. These genetic alterations were present in both cirrhotic and non-cirrhotic groups. By contrast, we did not find any beta-catenin gene alterations in the nine fibromellar carcinomas we examined. Nuclear accumulation of the protein was observed in 18 of them (25%). Remarkably, these included ten of the 14 tumors harboring somatic mutations in the beta-catenin gene (P < 0.001). Our results indicate that accumulation of beta-catenin resulting from genetic mutations is a frequent event in non-fibrolamellar type hepatocellular carcinoma. The close association between increased beta-catenin protein stability and mutation indicates that immunohistochemistry may be a powerful method for the detection of the mutated protein in future clinical practice.

Liver-specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion.

The high oncogenic efficiency of woodchuck hepatitis virus (WHV) has been correlated with the ability of this virus to provoke insertional activation of myc family genes. To assess the impact of viral integration on liver cell transformation, we have generated transgenic mice carrying the mutated c-myc gene and adjacent viral DNA from a woodchuck tumor, in original configuration. Virtually all mice from two different strains developed hepatocellular carcinoma with a mean latency period of 8-12 months. The c-myc transgene was expressed transiently in neonatal livers, and re-expressed at preneoplastic and neoplastic stages in adult livers. Woodchuck c-myc mRNA driven by the normal P1 and P2 promoters and WHV-specific transcripts encoding viral surface antigens were produced in a strictly co-regulated fashion during development and tumorigenesis, indicating a predominant regulatory influence of the viral enhancer. Furthermore, the activity of the viral enhancer in response to various biological stimuli was apparently modulated by glucose uptake and glucagon/insulin balance in differentiated hepatocytes. In this model, a viral integration event selected from a naturally occurring tumor proved to be determinant for induction of hepatocarcinogenesis, although enforced, liver-specific expression of c-myc was limited to a particular developmental stage.

Hepatocellular carcinoma in WHV/N-myc2 transgenic mice: oncogenic mutations of beta-catenin and synergistic effect of p53 null alleles.

The intronless N-myc2 gene was originally identified as the major target of hepatitis virus insertion in woodchuck liver tumors. Here we report that transgenic mice carrying the N-myc2 gene controlled by woodchuck hepatitis virus (WHV) regulatory sequences are highly predisposed to liver cancer. In a WHV/N-myc2 transgenic line, hepatocellular carcinomas or adenomas arose in over 70% of mice, despite barely detectable expression of the methylated transgene in liver cells. Furthermore, a transgenic founder carrying unmethylated transgene sequences succumbed to a large liver tumor by the age of two months, demonstrating the high oncogenicity of the woodchuck N-myc2 retroposon. Stabilizing mutations or deletions of beta-catenin were found in 25% of liver tumors and correlated with reduced tumor latency (P<0.05), confirming the important role of beta-catenin activation in Myc-induced tumorigenesis. The ability of the tumor suppressor gene p53 to cooperate with N-myc2 in liver cell transformation was tested by introducing a p53-null allele into WHV/N-myc2 transgenic mice. The loss of one p53 allele in transgenic animals markedly accelerated the onset of liver cancer (P=0.0001), and most tumors of WHV/N-myc2 p53+/Delta mice harbored either a deletion of the wt p53 allele or a beta-catenin mutation. These findings provide direct evidence that activation of N-myc2 and reduction of p53 levels act synergistically during multistage carcinogenesis in vivo and suggest that different genetic pathways may underlie liver carcinogenesis initiated by a myc transgene. Oncogene (2000).

Identification of homozygous deletions at chromosome 16q23 in aflatoxin B1 exposed hepatocellular carcinoma.

Loss of heterozygosity (LOH) represents the most frequent genetic alteration observed in hepatocellular carcinoma (HCC). Chromosome 16q is of particular interest as it exhibits LOH in 29% of HCC tumors and is frequently lost in breast, prostate, ovarian and gastric carcinomas. We genotyped 157 HCC tumors for 17 microsatellite markers distributed on chromosome 16q and determined a common region of LOH localized between the markers D16S518 and D16S504. By refining the boundaries of two interstitial LOH and two homozygous deletions, the critical region was delimited to 180 kb between D16S3096 and D16S3029. This region is located in intron 8 of the WWOX/FOR gene, but a search for mutations in all coding exons of this gene in 27 HCC tumors and cell lines did not reveal any tumor somatic alterations. Furthermore, by RT-PCR, no abnormal transcripts of this WWOX/FOR gene was detected in nine HCC cell lines. Finally, analysis of the p53 gene mutations with the clinical parameters of all tumors revealed that the two homozygous deletions have occurred in tumors presenting a R249S mutation. Our data revealed a relationship between chromosome 16q homozygous deletions and R249S p53 mutations in tumors where the patient had been exposed to aflatoxin B1 (P=0.002). These results are consistent with a role of aflatoxin B1 in the instability of chromosome 16q at the fragile site FRA16D. However, the nature of the specific gene that is altered during hepatocarcinogenesis remains to be elucidated.

No evidence of somatic FGFR3 mutation in various types of carcinoma.

Germline specific point mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3) are associated with autosomal dominant human skeletal dysplasia and craniosynostosis syndromes. Mutations identical to the germinal activating mutations found in severe skeletal dysplasias have been identified in certain types of cancer: at low frequency in multiple myeloma and cervix carcinoma and at high frequency in bladder carcinoma. We analysed, by SSCP and sequencing, the prevalence of FGFR3 mutations in 116 primary tumours of various types (upper aerodigestive tract, oesophagus, stomach, lung and skin). The regions analysed encompassed all FGFR3 point mutations previously described in severe skeletal dysplasia and cancers. No mutations were detected in the tumour types examined, suggesting that FGFR3 mutations are restricted to a few tumour types, the evidence to date suggesting that they are very specific to bladder carcinomas.

Right-sided endocarditis complicating peritoneovenous shunting for ascites.

We describe two patients with alcoholic cirrhosis in whom staphylococcal right-sided endocarditis developed after insertion of a peritoneovenous shunt (PVS). Massive pulmonary embolism caused early death in one patient. In the other patient, staphylococcal septicemia was cured after shunt removal and antibiotic treatment; recurrent endocarditis due to Corynebacterium xerosis ultimately caused the patient's death. No clinical manifestation of tricuspid valve dysfunction was noted in either patient, and right-sided endocarditis was recognized only at autopsy. The protracted contact of the tip of the venous line of PVS with the atrial wall is likely to be a major factor in the development of right-sided endocarditis in these patients.

In vitro chronic hepatic disease characterization with a multiparametric ultrasonic approach.

Although, high resolution, real-time ultrasonic (US) imaging is routinely available, image interpretation is based on grey-level and texture and quantitative evaluation is limited. Other potentially useful diagnostic information from US echoes may include modifications in tissue acoustic parameters (speed, attenuation and backscattering) resulting from disease development. Changes in acoustical parameters can be detected using time-of-flight and spectral analysis techniques. The objective of this study is to explore the potential of three parameters together (attenuation coefficient, US speed and integrated backscatter coefficient-IBC) to discriminate healthy and fibrosis subgroups in liver tissue. Echoes from 21 fresh in vitro samples of human liver and from a plane reflector were obtained using a 20-MHz central frequency transducer (6-30 MHz bandpass). The scan plane was parallel to the reflector placed beneath the liver. A 30 x 20 matrix of A-scans was obtained, with a 200-microm step. The samples were classified according to the Metavir scale in five different degrees of fibrosis. US speed, attenuation and IBC were estimated from standard methods described in the literature. Statistical tests were applied to the results of each parameter individually and indicated that it was not possible to identify all the fibrosis groups. Then a discriminant analysis was performed for the three parameters together resulting in a reasonable separation of fibrotic groups. Although the number of tissue samples is limited, this study opens the possibility of enhancing the discriminant capability of ultrasonic parameters of liver tissue disease when they are combined together.

Focal nodular hyperplasia of the liver: a comprehensive pathologic study of 305 lesions and recognition of new histologic forms.

Atypical histologic variants of focal nodular hyperplasia have been reported and are sometimes difficult to recognize. To characterize the morphologic spectrum of focal nodular hyperplasia, we studied 305 lesions surgically resected from 168 patients. Clinicomorphologic correlations were established by statistical analyses. The patients included 150 women and 18 men (sex ratio, 8:1; median age, 38 years). One hundred twenty-eight (76.2%) patients had solitary lesions, and 40 (23.8%) had 2 to 30 lesions. All 305 lesions measured 1 mm to 19 cm in diameter. Only 49% of these lesions had one to three macroscopic scars. Histologically, 245 (80.3%) lesions were of classical form, and 60 (19.7%) lesions were nonclassical. The latter were classified as focal nodular hyperplasia of telangiectatic form (47 lesions), of mixed hyperplastic and adenomatous form (five lesions), and with atypia of large cell type (eight lesions). Several benign or malignant tumors were found in association with these lesions. This large retrospective series of focal nodular hyperplasia shows the relative incidence of its classical and nonclassical forms. The absence of a central scar could explain the difficult preoperative diagnosis of some of the cases. The morphologic diagnostic criteria in this study require further prospective evaluation.

Impact and evolution of peliosis hepatis in renal transplant recipients.

Peliosis hepatis and hepatic sinusoidal dilatations are rare vascular liver diseases that occur at increased frequency in kidney transplant recipients. We retrospectively evaluated in kidney transplant recipients the natural history of vascular liver diseases, their impact on patient and graft survival, and the influence of AZA withdrawal. Between 1970 and 1990, vascular liver disease was diagnosed in 32 cadaver kidney transplant recipients 1-128 months after transplantation (mean 41 months). Diagnosis was based on histology in all cases. Patients received conventional immunosuppression (high dose steroids and AZA). Twenty patients had a minor form (sinusoidal dilatations or focal peliosis), while 12 had a major form (diffuse peliosis) of vascular hepatic disease. Two patients were lost to follow-up and 1 died at the time of diagnosis. In 12 patients (group 1), AZA dosage remained unchanged, while it was interrupted at the time of diagnosis in 17 patients (group 2). Five group 1 patients underwent serial liver biopsies, which showed persistence of vascular hepatic disease in 3 (with regenerative nodular hyperplasia in 1) and disappearance in 2 patients. Eight group 2 patients underwent serial liver biopsies, which showed disappearance of vascular hepatic disease in 6 patients and persistence in 2. Moreover, regenerative nodular hyperplasia was noted in 1 case, perisinusoidal fibrosis in 1 case, and cirrhosis in 6 cases. Three patients of group 1 and 11 patients of group 2 returned to dialysis a mean of 21 and 39 months after diagnosis, respectively. Eight patients died and death was clearly associated with major peliosis in 2 cases. In kidney transplant recipients, vascular hepatic disease may be associated with high mortality, especially in major forms. Our findings indicate that peliosis hepatis may lead to severe fibrosing liver lesions. The course of vascular hepatic disease is not clearly modified by AZA withdrawal.

Recurrence of Fabry's disease in a renal allograft eleven years after successful renal transplantation.

A case of Fabry's disease in a renal transplant recipient with a follow-up period of 11 years is reported. The patient suffered from renal, skin, peripheral nerve lesions, and asymptomatic cardiomegaly. Fabry's disease symptoms disappeared after transplantation. Improvement of renal function was rapidly observed, and it remained satisfactory during the whole posttransplantation period. The patient died of a severe, uncontrolled infection and of biliary peritonitis. Autopsy showed a polyvisceral accumulation of sphingolipids deposits. The engrafted kidney was histologically free of disease. Ultrastructurally, it revealed numerous sphingolipid inclusions in the endothelial cells of capillaries. The explanation of this complication could be attributed to: (1) high circulating levels of plasma substrates locally overwhelming the enzymatic capability of the graft endothelial cells; and (2) the endothelial cells originated from the recipient but not from the donor, an occurrence that has been described after transplantation. Rejection and the newly formed deposits in the endothelial cells may lead to the loss of the engrafted organ. As a consequence of the increasing possibility of organ transplantation, this complication should be detected by studying the blood vessels ultrastructurally in order to evaluate the condition of the transplant.

Fulminant hepatitis in renal transplant recipients. The role of the delta agent.

In a series of about 300 renal transplant patients followed from 1972 to 1983, 3 cases of fulminant hepatitis were observed in HBs Ag-positive patients. In the liver biopsies of 2/3 of them, the delta antigen was detected by direct immunofluorescence with a specific anti-delta serum. This result demonstrates the responsibility of the delta agent for the development of fulminant hepatitis, and emphasizes the possibility of delta infection in HBs Ag-positive transplant patients with severe hepatitis.

Is renal transplantation involved in post-transplantation liver disease? A prospective study.

Various lesions of the liver commonly observed in renal transplant recipients are usually considered as a consequence of the transplantation procedures (immunosuppression, drug toxicity, alteration of immune responses to various viruses). A group of 64 patients all treated with corticosteroids and azathioprine was studied prospectively, and serial liver biopsies were performed on the day of transplantation and at 1 and 3 years after transplantation. Chronic hepatitis was already present in 40% of the patients on the day of transplantation and an increase of only 15% in the frequency of this condition was observed 3 years later. The presence of HBsAg in 45% of the patients at the time of transplantation was significantly associated with liver lesions. In about 3% of the cases, transplantation was directly responsible for a liver disease (peliosis hepatitis). During the followup period an evolution from chronic persistent hepatitis to chronic active hepatitis was observed with an abnormally high frequency (25%). We conclude that most of the liver diseases observed in transplant recipients are the consequence of events before transplantation and probably related to hemodialysis.

Studies of portal hemodynamics and hepatic oxygen consumption during acute liver allograft rejection.

Hemodynamics and oxygen variables, plasma cytokines, and histological features of a liver tissue sample obtained by transvenous biopsy were evaluated during 65 episodes of acute rejection. The hepatic venous pressure gradient was significantly higher in patients with acute rejection than in those without (5.1+/-0.3 vs. 3.1+/-0.2 mmHg, P<0.01). The increase in pressure gradient was related to the severity of rejection lesions. Hepatic blood flow was significantly lower in patients with than in those without acute graft rejection (1.28+/-0.11 vs. 1.75+/-0.13 L/min, P<0.05). Plasma interleukin-6 levels were significantly increased in patients with acute rejection and positively correlated with pressure gradient values. In patients with acute rejection, a significant decrease in hepatic venous oxygen content (-16%) was associated with a significant increase in hepatic oxygen consumption (+24%), whereas hepatic oxygen transport did not change significantly. In treated patients with a favorable response, the pressure gradient decreased significantly by 46%, but it remained elevated in patients who later developed chronic graft rejection. In conclusion, this study confirms that acute graft rejection may induce an increase in portal pressure, which is related to the severity of rejection lesions. It also shows that acute rejection decreases hepatic blood flow and increases hepatic oxygen consumption. In addition, it suggests that the hepatic venous pressure gradient might be useful to determine the outcome of rejection.

Metabolic activation of the tricyclic antidepressant amineptine--II. Protective role of glutathione against in vitro and in vivo covalent binding.

Incubation of [11-14C]amineptine (1 mM) with an NADPH-generating system and hamster liver microsomes resulted in the in vitro covalent binding of an amineptine metabolite to microsomal proteins; this binding was decreased by 41-71% in the presence of cysteine, lysine, glycine or glutathione (0.5 mM). An inverse relationship was found between the concentration of glutathione in the incubation mixture (0.25-4 mM) and the extent of covalent binding in vitro, which became undetectable at concentrations of glutathione of 2 mM and higher. Administration of [11-14C]amineptine (300 mg/kg-1 i.p.) to hamsters pretreated with phorone (500 mg/kg i.p.) resulted in the in vivo covalent binding of an amineptine metabolite to hepatic proteins. This binding was increased by phenobarbital-pretreatment and decreased by piperonyl butoxide-pretreatment. After various doses of phorone (150-500 mg/kg), an inverse relationship was found between hepatic glutathione content and in vivo covalent binding. Administration of amineptine alone (300 mg/kg i.p.) depleted hepatic glutathione by 16% only; in these animals, in vivo covalent binding was undetectable from background. Amineptine (300 mg/kg i.p.) did not produce hepatic necrosis, even in hamsters pretreated with phorone and/or phenobarbital. We conclude that physiologic concentrations of glutathione essentially prevent the in vivo covalent binding of an amineptine metabolite to hepatic proteins, and that this binding does not produce liver cell necrosis in hamsters.

Protective effect of 16,16-dimethyl prostaglandin E2 on the hepatotoxicity of bromobenzene in mice.

It has been suggested that 16,16-dimethyl prostaglandin E2 may have a cytoprotective effect in the liver. To assess this hypothesis, we determined the effects of this prostaglandin on the metabolism and toxicity of bromobenzene in mice. Administration of 16,16-dimethyl prostaglandin E2 (50 micrograms/kg s.c., 30 min before, and every 6 hr after, the administration of bromobenzene) did not modify the disappearance curves of unchanged bromobenzene from plasma and liver, and did not modify the amount of bromobenzene metabolites covalently bound to hepatic proteins 1-24 hr after the administration of a toxic dose of bromobenzene (0.36 ml/kg i.p.). The prostaglandin, however, markedly reduced serum alanine aminotransferase activity, the extent of liver cell necrosis, the depletion of glutathione, and the disappearance of cytochrome P-450 after administration of this toxic dose of bromobenzene (0.36 ml/kg i.p.). It also markedly reduced mortality after administration of a lethal dose of bromobenzene (0.43 ml/kg i.p.). We conclude that 16,16-dimethyl prostaglandin E2 can prevent hepatic necrosis without decreasing the covalent binding of bromobenzene metabolites to hepatic proteins. The mechanism for this dissociation between covalent binding and toxicity remains unknown.

Mechanism for the protective effects of silymarin against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Evidence that silymarin acts both as an inhibitor of metabolic activation and as a chain-breaking antioxidant.

Administration of silymarin (800 mg/kg i.p.) 30 min before carbon tetrachloride (18 microL/kg i.p.) did not modify total hepatic levels of CCl4 and metabolites in mice, but decreased by 40% the in vivo covalent binding of CCl4 metabolites to hepatic lipids at 2 hr. This pretreatment decreased by 60% the exhalation of ethane during the first hour after CCl4, and decreased by 50% the incidence of liver cell necrosis. In vitro, silymarin (800 micrograms/mL) decreased by 50 to 70% various monooxygenase activities, and decreased by 20% the covalent binding of CCl4 metabolites to microsomal proteins. Silymarin (800 micrograms/mL) decreased by 70% in vitro lipid peroxidation mediated by CCl4 metabolites, and decreased by 90% peroxidation mediated by NADPH alone. Silibinin, one of the three isomers composing silymarin, also decreased carbon tetrachloride-induced lipid peroxidation; this effect, however, was less than that of silymarin in vitro, and was more transient in vivo. Pretreatment with silibinin (800 mg/kg i.p.) 30 min before CCl4 (18 microL/kg i.p.) did not improve SGPT activity or liver histology at 24 hr. We conclude that silymarin prevents carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice, firstly, by decreasing the metabolic activation of CCl4, and, secondly, by acting as a chain-breaking antioxidant.

Metabolic activation of the antidepressant tianeptine. II. In vivo covalent binding and toxicological studies at sublethal doses.

Administration of [14C]tianeptine (0.5 mmol/kg i.p.) to non-pretreated hamsters resulted in the in vivo covalent binding of [14C]tianeptine metabolites to liver, lung and kidney proteins; this very high dose (360-fold the human therapeutic dose) depleted hepatic glutathione by 60%, and increased SGPT activity 5-fold. Lower doses (0.25 and 0.125 mmol/kg) depleted hepatic glutathione to a lesser extent and did not increase SGPT activity. Pretreatment of hamsters with piperonyl butoxide decreased in vivo covalent binding to liver proteins, and prevented the increase in SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). In contrast, pretreatment of hamsters with dexamethasone increased in vivo covalent binding to liver proteins, and increased SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). Nevertheless, liver cell necrosis was histologically absent 24 hr after the administration of tianeptine (0.5 mmol/kg i.p.) to non-pretreated or dexamethasone-pretreated hamsters. In vivo covalent binding to liver proteins also occurred in mice and rats, being increased by 100% in dexamethasone-pretreated animals. In vivo covalent binding to liver proteins was similar in untreated female Dark Agouti rats and in female Sprague-Dawley rats. These results show that tianeptine is transformed in vivo by cytochrome P-450, including glucocorticoid-inducible isoenzymes, into chemically reactive metabolites that covalently bind to tissue proteins. The metabolites, however, exhibit no direct hepatotoxic potential in hamsters below the sublethal dose of 0.5 mmol/kg i.p. The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

The drug methoxsalen, a suicide substrate for cytochrome P-450, decreases the metabolic activation, and prevents the hepatotoxicity, of carbon tetrachloride in mice.

Methoxsalen, a potent suicide inhibitor of cytochrome P-450 that can be used in humans, might be of value for the prevention of hepatitis in subjects with carbon tetrachloride poisoning. As a preliminary step, we have determined its effects on the hepatotoxicity of carbon tetrachloride in mice. Several monooxygenase activities, the in vitro covalent binding of carbon tetrachloride metabolites to microsomal proteins, and in vitro microsomal lipid peroxidation initiated by carbon tetrachloride metabolites were decreased by 60-90% in microsomes from mice killed 2 hr after the administration of methoxsalen (250 mumol X kg-1); microsomal lipid peroxidation mediated by endogenous iron and NADPH was not modified. Administration of methoxsalen (250 mumol X kg-1) 30 min before carbon tetrachloride (0.1 ml X kg-1) decreased both the in vivo formation of conjugated dienes in microsomal lipids and the in vivo covalent binding of carbon tetrachloride metabolites to lipids and proteins. This pretreatment completely prevented the hepatotoxicity of carbon tetrachloride. Other cytochrome P-450 inhibitors (cimetidine, SKF 525-A or piperonyl butoxide) given at this low molar dose (250 mumol X kg-1) exerted no protective effect. Methoxsalen (500 mumol X kg-1) was also effective, but only partially, when given 30 min after carbon tetrachloride (0.025 ml X kg-1). We conclude that pretreatment with methoxsalen decreases the metabolic activation of carbon tetrachloride, and completely prevents its hepatotoxicity in mice. Post-treatment with methoxsalen must be given early and is only partially effective in mice.