Previous work has shown a reduction in cephaloridine nephrotoxicity in a diabetic rat model. The following studies examined in vitro cephaloridine toxicity in renal slices from normoglycemic and diabetic Fischer 344 rats. Diabetes was induced by acute intraperitoneal injection of 35 mg/kg streptozotocin. Renal cortical slices were isolated from normoglycemic and diabetic animals. Tissues were exposed to 0-5 mM cephaloridine for 15-120 min. Pyruvate-directed gluconeogenesis was diminished in all groups exposed to 2-5 mM cephaloridine for 60-120 min. Leakage of lactate dehydrogenase (LDH) was apparent only in the normoglycemic group in the presence of 4-5 mM cephaloridine for 120 min. LDH leakage was not increased at any cephaloridine concentration in the diabetic tissue. Total glutathione levels were compared in renal cortical slices exposed to cephaloridine for 30-120 min. Baseline values for glutathione were comparable between normoglycemic and diabetic tissue suggesting that the mechanism for reduced toxicity was not due to higher glutathione levels in diabetic tissue. Total glutathione levels were diminished more rapidly in normoglycemic than diabetic tissue by incubation with 5 mM cephaloridine. Comparison of cephaloridine accumulation indicated that diabetic tissue accumulated less cephaloridine than the normoglycemic group when tissues were incubated with 0-2 mM cephaloridine. However, renal slice accumulation was similar between normoglycemic and diabetic groups following in vitro incubation with 4-5 mM cephaloridine. These results suggest that the mechanism for reduced in vitro cephaloridine toxicity in diabetic tissue cannot be limited to differences in accumulation and must include an unidentified cellular component.
Metabolism of dichloromethane (DCM) to formaldehyde (HCHO) via a glutathione S-transferase (GST) pathway is thought to be required for its carcinogenic effects in B6C3F1 mice. In humans, this reaction is catalyzed primarily by the protein product of the gene GSTT1, a member of the Theta class of GST, and perhaps to a small extent by the protein product of the gene GSTM1. Humans are polymorphic with respect to both genes. Since HCHO may bind to both DNA and RNA forming DNA-protein crosslinks (DPX) and RNA-formaldehyde adducts (RFA), respectively, these products were determined in isolated hepatocytes from B6C3F1 mice, F344 rats, Syrian golden hamsters, and humans to compare species with respect to the production of HCHO from DCM and its reaction with nucleic acids. Only mouse hepatocytes formed detectable amounts of DPX, the quantities of which corresponded well with quantities of DPX formed in the livers of mice exposed to DCM in vivo [Casanova, M., Conolly, R.B., and Heck, H. d'A. (1996). Fundam. Appl. Toxicol. 31, 103-116]. Hepatocytes from all rodent species and from humans with functional GSTT1 and GSTM1 genes formed RFA. No RFA were detected in human cells lacking these genes. Yields of RFA in hepatocytes of mice were 4-fold higher than in those of rats, 7-fold higher than in those of humans, and 14-fold higher than in those of hamsters. The RFA:DPX ratio in mouse hepatocytes incubated with DCM was approximately 9.0 +/- 1.4, but it was 1.1 +/- 0.3 when HCHO was added directly to the medium, indicating that HCHO generated internally from DCM is not equivalent to that added externally to cells and that it may occupy separate pools. DPX were not detected in human hepatocytes even at concentrations equivalent to an in vivo exposure of 10,000 ppm; however, the possibility that very small amounts of DPX were produced from DCM cannot be excluded, since HCHO was formed in human cells. Maximal amounts of DPXliver that might be formed in humans were predicted from the amounts in mice and the relative amounts of RFA in hepatocytes of both species. With predicted DPXliver as the dosimeter, the unit risk, the upper 95% confidence limit on the cancer risk, and the margin of exposure were calculated at several concentrations using the linearized multistage and benchmark dose methods. Since the actual delivered dose is smaller than that predicted, the results suggest that DCM poses at most a very low risk of liver cancer to humans.
In vitro assessment of human lymphocyte viability by trypan blue dye exclusion in the presence of an external metabolizing system (microsomes plus NADPH) has been shown to be a useful method in assessing predisposition to idiopathic toxicity in response to various anticonvulsant drugs. The trypan blue method, however, is labor intensive, is time consuming, is prone to human error, is not suitable for high-volume toxicity screening, and excludes autolysed cells. The objective of this study was to develop a rapid, high-capacity, objective, and easy in vitro cytotoxicity method for the detection of metabolism-dependent cytotoxicity of a test chemical. The in vitro system uses an external metabolizing system (rabbit microsomes) in conjunction with isolated human lymphocytes as the target cells. Cellular toxicity was determined by assessing plasma membrane integrity using a membrane-impermeant fluorescent nucleic acid dye (YO-PRO-1) and a multiwell plate scanner for fluorescence. Using this system, cells incubated with either acetaminophen (1500 micrograms/ml), carbamazepine (62.5 microM), phenytoin (62.5 microM), or phenobarbital (62.5 microM) showed net increases in percentage cell death of 31 +/- 5, 11 +/- 4, 0 +/- 3, and 2 +/- 3, respectively. A metabolism-dependent concentration-response was observed for valproic acid-induced cytotoxicity, which approached a plateau at a concentration of 4000 micrograms/ml with a net percentage cell death of 31 +/- 4. This technique resolves various technical difficulties inherent in viability determinations by the trypan blue exclusion method. The YO-PRO-1 method also may be useful in a clinical setting for the assessment of patients with a genetically determined susceptibility to certain drugs and for identifying the responsible drug in patients with idiopathic toxicity undergoing multiple-drug therapy.
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