Structure-activity relationship for the intrinsic hepatotoxicity of dinitrotoluenes.

The relation of various structural parameters to hepatotoxic potential was investigated by using six dinitrotoluene (DNT) isomers and isolated rat hepatocyte suspensions as the biological test system. DNT-induced hepatotoxicity was found to correlate with an inhibition of protein synthesis and an increase in lactate dehydrogenase (LDH) release but not with lipid peroxidation. With each isomer, protein synthesis inhibition was the most sensitive indicator of cytotoxicity. Regardless of the indicator, ortho- and para-substituted isomers were more hepatotoxic at the same concentration than meta-substituted isomers. High-performance liquid chromatograms (HPLC) on samples at 4 h revealed significant quantities of reduced metabolites in the medium. However, increased lipid peroxidation (formation of thiobarbituric acid reactants or evolution of ethane) in the cells was not consistently demonstrated. log EC50 for protein synthesis inhibition and log EC20 for LDH release were linearly correlated with the C atomic charge on the ring carbons bearing the nitro substituents by using molecular orbital (MNDO calculations) theory. The relation was used to predict the hepatotoxic potentials of untested nitrotoluenes, and the predictions were verified to a first approximation by using three trinitrotoluene isomers.

Application of a hepatocyte-erythrocyte coincubation system to studies of cyanide antidotal mechanisms.

A coincubation system composed of hepatocytes in primary monolayer culture and erythrocytes suspended in the culture medium was developed and used as a model for investigations of mechanisms of cyanide antidote action at the cellular level. Hepatocyte ATP was used as the cytotoxicity indicator. Treatment of rat hepatocytes in the coincubation system with KCN (1.0 mM) for 10 min at 37 degrees C selectively reduced hepatocyte ATP levels to 33 +/- 15% of control (no KCN added) levels. 4-dimethylaminophenol (DMAP), cobalt(II) chloride, sodium nitrite, sodium thiosulfate, or a combination of the last two antidotes added to the KCN-containing medium significantly reversed ATP depression and the response was concentration dependent. The relative effectiveness, on a molar basis, was estimated to be DMAP greater than CoCl2 much greater than NaNO2 congruent to Na2S2O3. NaNO2 and DMAP induced methemoglobin formation in the absence of cyanide and cyanmethemoglobin formation in its presence; erythrocytes were required in the medium for effectiveness. CoCl2 produced neither cyanmethemoglobin nor thiocyanate in appreciable quantities nor required erythrocytes for antagonism. Na2S2O3 converted cyanide to thiocyanate and reversed ATP depression without erythrocytes in the medium. The addition of erythrocytes increased these rates significantly and to a greater extent than albumin. The overall results are consistent with previously proposed modes of action for these antidotes. However, the enhancement in cyanide metabolism and ATP recovery with Na2S2O3 and erythrocytes in the system was unexpected and raises the possibility that erythrocytes may contribute to cyanide disposition and antagonism in vivo when this antidote is administered.

Comparison of amphetamine metabolism using isolated hepatocytes from five species including human.

Isolated hepatocyte suspensions from rat, rabbit, dog, squirrel monkey and human livers were used to study the metabolism of amphetamine (AMP), a drug for which species-dependent differences in metabolism have been demonstrated in vivo. Hepatocytes were isolated by perfusion of the whole liver or of biopsy specimens. In general, the metabolite profile of hepatocytes from each species corresponded to the profile of urinary metabolites identified previously. Rat hepatocytes primarily metabolized AMP by aromatic hydroxylation to p-hydroxyamphetamine. Rabbit hepatocytes converted AMP almost exclusively to products of the oxidative deamination pathway. Metabolism by hepatocytes from the other three species was mixed, but oxidative deamination was somewhat more active than aromatic hydroxylation in dog, squirrel monkey and human hepatocytes. The overall rate of AMP metabolism differed significantly among the species; the half-life in the hepatocyte suspensions varied about 70-fold, with rabbit less than rat less than dog less than squirrel monkey = human. Metabolism of AMP by human hepatocytes mare closely resembled metabolism by squirrel monkey liver cells than the other species in terms of metabolite profile and rate. However, the disposition of phenylacetone, a product of oxidative deamination of AMP, varied in hepatocytes from the two primate species. Thus, the metabolism of AMP by isolated hepatocytes was unique for each species examined. These studies demonstrate the applicability of isolated hepatocytes to the study of interspecies differences in hepatic xenobiotic metabolism, providing an in vitro technique that can be readily adapted to human liver tissue.

Biological evaluation of some ionophore-polymeric chelator combinations for reducing iron overload.

The potential efficacy of drug combinations that employ an ionophore to mobilize excess hepatic Fe and transport it to the gut for chelation to nonabsorbable polymers and excretion was evaluated in three ways: 1) two-phase partition experiments to assess the ionophoretic capability of lipid-soluble Fe(III)-chelators; 2) in vitro screening with Fe-loaded hepatocyte cultures; and 3) evaluation in Fe-dextran-loaded mice given a low-Fe-containing diet for maximal sensitivity. In the partition experiments, desferal (DF), (desferri)ferrichrome, pyridoxyl isonicotinoyl acid hydrazone hydrochloride (PINH), 2,3-dihydroxybenzoic acid, cholylhydroxamic acid and caffeic acid were effective to varying degrees in transferring Fe(III) from an aqueous to an organic hydrophobic phase. PINH, the most effective, combined with poly[vinylamine-vinyl(2,3-dihydroxybenzenecarboxamide)-vinyl sulfonate sodium salt] (DHBP), increased net 59Fe release from Fe-loaded hepatocytes above the sum of that released by each drug alone and comparable with DF-induced levels. In the mouse bioassay, the combination of PINH (8.1 mg/kg) and DHBP (170 mg/kg 2 times daily) caused a 32% increase in fecal Fe output, comparable with total Fe excretion with DF, but higher doses were toxic and lower doses ineffective. Furthermore, less than 5% of the total Fe load administered was excreted during the 4-day treatment period by either DF or the PINH-DHBP drug combination.

Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion.

Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals.