Jack E. Dabbs

Primary active transport of organic anions on bile canalicular membrane in humans

Biliary excretion of several anionic compounds was examined by assessing their ATP-dependent uptake in bile canalicular membrane vesicles (CMV) prepared from six human liver samples. 2, 4-Dinitrophenyl-S-glutathione (DNP-SG), leukotriene C4 (LTC4), sulfobromophthalein glutathione (BSP-SG), E3040 glucuronide (E-glu), beta-estradiol 17-(beta-D-glucuronide) (E2-17G), grepafloxacin glucuronide (GPFXG), pravastatin, BQ-123, and methotrexate, which are known to be substrates for the rat canalicular multispecific organic anion transporter, and taurocholic acid (TCA), a substrate for the bile acid transporter, were used as substrates. ATP-dependent and saturable uptake of TCA, DNP-SG, LTC4, E-glu, E2-17G, and GPFXG was observed in all human CMV preparations examined, suggesting that these compounds are excreted in the bile via a primary active transport system in humans. Primary active transport of the other substrates was also seen in some of CMV preparations but was negligible in the others. The ATP-dependent uptake of all the compounds exhibited a large inter-CMV variation, and there was a significant correlation between the uptake of glutathione conjugates (DNP-SG, LTC4, and BSP-SG) and glucuronides (E-glu, E2-17G, and GPFXG). However, there was no significant correlation between TCA and the other organic anions, implying that the transporters for TCA and for organic anions are different also in humans. When the average value for the ATP-dependent uptake by each preparation of human CMVs was compared with that of rat CMVs, the uptake of glutathione conjugates and nonconjugated anions (pravastatin, BQ-123, and methotrexate) in humans was approximately 3- to 76-fold lower than that in rats, whereas the uptake of glucuronides was similar in the two species. Thus there is a species difference in the primary active transport of organic anions across the bile canalicular membrane that is less marked for glucuronides.Biliary excretion of several anionic compounds was examined by assessing their ATP-dependent uptake in bile canalicular membrane vesicles (CMV) prepared from six human liver samples. 2,4-Dinitrophenyl- S-glutathione (DNP-SG), leukotriene C4(LTC4), sulfobromophthalein glutathione (BSP-SG), E3040 glucuronide (E-glu), β-estradiol 17-(β-d-glucuronide) (E2-17G), grepafloxacin glucuronide (GPFXG), pravastatin, BQ-123, and methotrexate, which are known to be substrates for the rat canalicular multispecific organic anion transporter, and taurocholic acid (TCA), a substrate for the bile acid transporter, were used as substrates. ATP-dependent and saturable uptake of TCA, DNP-SG, LTC4, E-glu, E2-17G, and GPFXG was observed in all human CMV preparations examined, suggesting that these compounds are excreted in the bile via a primary active transport system in humans. Primary active transport of the other substrates was also seen in some of CMV preparations but was negligible in the others. The ATP-dependent uptake of all the compounds exhibited a large inter-CMV variation, and there was a significant correlation between the uptake of glutathione conjugates (DNP-SG, LTC4, and BSP-SG) and glucuronides (E-glu, E2-17G, and GPFXG). However, there was no significant correlation between TCA and the other organic anions, implying that the transporters for TCA and for organic anions are different also in humans. When the average value for the ATP-dependent uptake by each preparation of human CMVs was compared with that of rat CMVs, the uptake of glutathione conjugates and nonconjugated anions (pravastatin, BQ-123, and methotrexate) in humans was ∼3- to 76-fold lower than that in rats, whereas the uptake of glucuronides was similar in the two species. Thus there is a species difference in the primary active transport of organic anions across the bile canalicular membrane that is less marked for glucuronides.

Metabolism and cytotoxicity of acetaminophen in hepatocytes isolated from resistant and susceptible species

Acetaminophen (APAP) disposition was studied in vitro using hepatocytes isolated from rats, hamsters, rabbits, and dogs, species that vary markedly in susceptibility to the hepatotoxicity of this drug. Metabolism was assessed by concurrent measurements of glutathione depletion and protein adduct formation (activation pathway) and of total aqueous metabolite production (detoxication pathways). Cytotoxicity was monitored by cell count and by lactate dehydrogenase (LDH) release to culture medium. In agreement with whole animal studies, hepatocytes from hamsters were very susceptible to APAP-induced toxicity whereas rat and rabbit hepatocytes were resistant. In vivo data were unavailable for the dog, but dog hepatocytes were also relatively resistant. Parameters of APAP metabolism generally correlated with the species susceptibility ranking; however, no single parameter was an ideal index of the sensitivity observed. As predicted by the cytotoxicity data, hamster hepatocytes produced more covalent adducts of APAP, were depleted of GSH more rapidly, and detoxified APAP by formation of polar metabolites at a slower rate than rat hepatocytes. On the other hand, rabbit hepatocytes had no detectable covalent adducts, retained higher amounts of GSH, and metabolized more APAP to polar conjugates than the other species. Dog hepatocytes formed low amounts of both covalent adducts and conjugates. These studies indicate that interspecies comparisons using isolated hepatocytes to study xenobiotic metabolism and the resulting cytotoxicity are feasible, but for a clear understanding of observed differences, it is necessary to study the interrelationships between the toxication and detoxication pathways of metabolism.

Oxidative DNA damage induced by potassium bromate in isolated rat renal proximal tubules and renal nuclei

Oxidative damage caused by potassium bromate (KBrO3), a rat renal carcinogen, was investigated using in vitro preparations of rat renal proximal tubules (RPT) and renal nuclear fractions. Release of lactate dehydrogenase and decrease of SH-group content in RPT (1 mg protein/ml) by KBrO3 (0.5-5 mM) in a concentration- and time-dependent manner were observed. Peroxidized arachidonic acid and 8-hydroxydeoxyguanosine (8-OH-dG) levels in RPT were increased after administration of 2 and 5 mM KBrO3. 8-OH-dG formation was observed after incubation of renal nuclei with a lipid-peroxiding system, autooxidized methyl linolenate, or KBrO3. These findings provide support for involvement of lipid peroxidation in producing oxidized DNA damage by KBrO3 directly to RPT, the target site for renal carcinogenesis.

Functional integrity of isolated rat hepatocytes prepared by whole liver vs biopsy perfusion.

Isolated rat hepatocytes prepared by a newly developed, versatile biopsy perfusion method (HB cells) were compared with hepatocytes prepared by conventional whole liver perfusion (HP cells), immediately after isolation and after 6 h in suspension culture. Thirteen parameters were used to assess the functional integrity of these cells. Both methods produced high yields of metabolically active hepatocytes that were virtually indistinguishable from each other. After 6 h, the average viability of both cell isolates declined approximately 10%, mixed function oxidase activities were decreased approximately 25% at most, and GSH levels were actually increased; other parameters were not significantly changed. The data indicate that HB-cell isolates are at least as viable and metabolically active as HP cells, and, because the biopsy perfusion method can be applied to liver samples from any species, it facilitates comparative studies.

Effects of soya bean flakes and liquorice root extract on enzyme induction and toxicity in B6C3F1 mice.

Both soya bean flakes (SBF) and liquorice root extract (LRE) have previously been reported to have anticarcinogenic properties, which have been thought to be related to an increased activity of specific enzymes responsible for the detoxification of chemical carcinogens. 30- and 90-day studies were conducted in male B6C3F1 mice to determine which, if any, of several detoxification enzymes are induced by SBF or LRE. Mice fed 8 and 25% LRE showed a variety of adverse clinical signs, poor weight gain and 30% mortality. Significant increases in liver:body weight ratios were observed in both the SBF and LRE groups. No significant treatment-related gross autopsy findings were observed in any of the SBF groups. A number of abnormalities were observed in the LRE groups, including lesions of the kidney, liver, spleen and thymus. Liver samples from the 90-day study were analysed for 7-ethoxycoumarin O-deethylase (7-ECOD), benzo[a]pyrene hydroxylase (BPH), superoxide dismutase (SOD), glutathione S-transferase (GST) and UDP-glucuronyl transferase (UDPGT) at 90 days, and at an interim 30-day autopsy. No treatment-related increases were observed for BPH or SOD. Both SBF and LRE induced modest increases in UDPGT activity. SBF induced modest increases in GST activity, but LRE decreased this activity. 7-ECOD activity was significantly increased by LRE and decreased by SBF. Samples from a 30-day study in which both LRE and SBF were administered at various dose levels were examined for UDPGT activity; all dose groups showed decreases in UDPGT activity relative to controls. The results suggest that both SBF and LRE may alter the activities of specific enzymes involved in the detoxification of chemical carcinogens; however, the combination of these two foodstuffs may not produce an additive effect in B6C3F1 mice.

Characterization of Isolated Renal Proximal Tubules for Nephrotoxicity Studies

Isolated tubule or cell suspensions offer an important way of studying the mechanisms of nephrotoxicity in vitro, and screening novel compounds for their potential adverse affects on the kidney. The major drawback, however, appeared to be the short in vitro lifespan of isolated tubules prepared by any technique. In general, most investigators limit incubations to no more than 1 or 2 hr because of loss of viability and functional capabilities. Ormstad (1984) reported rapid loss of viability of isolated renal tubules and cells, where greater than 25% of the cellular lactate dehydrogenase (LDH) leaked to the medium in 1.5 to 2 hr of incubation. Obatomi and Plummer (1986) observed a 40% loss in tubule cell viability during 3-hr incubations of rat proximal tubules. Loss of renal functional capabilities, such as O2 consumption, has also been reported for isolated tubules (Harris et al., 1981).

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.

Stress initiated during isolation of rat renal proximal tubules limits in vitro survival.

The effects of oxidative damage were assessed in rat proximal tubule fragments (isolated by collagenase perfusion) by monitoring lactate dehydrogenase release (LDH-R) to measure cell viability and thiobarbituric acid (TBA) reactive material to follow oxidative damage. Increasing the oxygen content in the incubation atmosphere from 10 to 95% significantly increased LDH-R and TBA reactants. Addition of butylated hydroxytoluene or deferoxamine (DF) to the medium prevented these changes, but ascorbic acid or mannitol had no positive effect. Lima bean trypsin inhibitor also reduced LDH leakage significantly when added to the medium, but not when added to the perfusion buffers. In contrast, adding DF to the perfusate during tubule isolation produced the most pronounced benefit; net LDH-R after 4 hr was about 10% in tubules prepared this way compared to 20% when DF was omitted. Basal oxygen consumption declined to approximately the same extent as LDH-R increased. Maintenance of nystatin-stimulated respiration, ATP/ADP, GSH content and total adenine nucleotides indicated good cell function. These results suggest that oxidative damage initiated during the tubule isolation procedure limits cell survival but this effect can be counteracted substantially by the addition of DF to the perfusion buffer.

Biodistribution of the Multidentate Hydroxypyridinonate Ligand [14C]‐3,4,3‐LI(1,2‐HOPO), a Potent Actinide Decorporation Agent

Preclinical Research

Studies of nephrotoxic agents in an improved renal proximal tubule system

Renal proximal tubule fragments (RPT) were prepared from young-adult, male F-344 rats by deferoxamine/collagenase perfusion and evaluated as a potential model for mechanistic studies and screening, using known nephrotoxins. Chloroform and S-(1,2- dichlorovinyl )- l - cysteine (DCVC) produced depressed O(2) consumption rates (basal and/or nystatin-stimulated) and lactate dehydrogenase (LDH) release during 8-hr incubations at 0.5 mg RPT protein/ml. Cytochrome P-450 inhibitors piperonyl butoxide and metyrapone were either without effect or potentiated chloroform-induced toxicity. DCVC was more cytotoxic to RPT than to rat hepatocytes. The cytotoxic potency for cephalothin relative to cefazolin decreased as RPT content in the medium was increased to 3.0 mg protein/ml, giving a rank order more in accord with results reported in vivo. Cephalosporins markedly depressed brush border alkaline phosphatase (ALP) activity, without affecting gamma-glutamyltranspeptidase activity; the effect on ALP was less sensitive to the RPT level. Acetaminophen (25 mm) and p-aminophenol (1.0 mm) induced LDH release without ALP depression and inhibited mitochondrial respiration. These results in general corresponded well with in vivo responses and indicate that this RPT system may be valuable for studies of chemical-induced nephrotoxicity.