Ruth E. Billings

l-Acetylmethadol (LAM) treatment of opiate dependence: plasma and urine levels of two pharmacologically active metabolites.

The metabolic conversion of α-l-acetylmethadol (LAM) to α-l-noracetylmethadol (NAM) and α-l-dinoracetylmethanol (NNAM), has been studied in three opiate addicts being maintained on 100 mg of LAM three times weekly. Plasma levels of NAM and NNAM were established shortly after the initial dose of LAM. The plasma level of NNAM was substantially higher following repeated dosing than following the initial dose. The combined daily urinary excretion of LAM, NAM and NNAM was 6–8 times greater after repeated dosing than after the initial dose. Since NAM and NNAM, which are formed by the sequential N-demethylation of LAM are both considerably more active morphine-like agonists than is LAM itself, it is likely that the pharmacological effects of LAM are due to NAe NAM and NNAM. Variations in the rates of formation and elimination of NAM and NNAM may partially explain the variability in response seen in LAM maintenance therapy.

Interlaboratory validation of a new assay for DNA-protein crosslinks

In 1992, a simple and sensitive assay for detecting DNA-protein crosslinks was developed [1]. In an effort to facilitate the greater use of the assay, a number of studies were conducted to evaluate its reliability and reproducibility. During this work, the assay was used to assess whether various metals and other compounds could induce crosslinks in cultured human lymphocytes (Epstein-Barr virus-transformed Burkitts Lymphoma cell line). Potassium permanganate, mercury chloride, lead nitrate, magnesium perchlorate, aluminum chloride, and cadmium chloride did not induce DNA-protein crosslinks at either cytotoxic or non-cytotoxic levels. Copper sulfate, arsenic trioxide, and potassium chromate induced DNA-protein crosslinks only at cytotoxic concentrations. Acute lethality of the cells was assessed immediately after exposure to metals by trypan blue exclusion while long-term lethality was assessed by cell proliferation and trypan blue exclusion following an incubation period of 5 days after exposure to the metal compound. All metals exhibited more toxicity in the long-term lethality assay compared to the short-term assay. The cultured human lymphocytes treated with various doses of lead acetate, cadmium chloride, arsenic trioxide and copper sulfate, as well as cis-platinum and chromate, were sent to four different laboratories to compare the reliability and reproducibility of the DNA-protein crosslink assay. Depending on the chemical studied, there were quantitative differences in the results observed among the various laboratories using the assay. However, all laboratories generally showed that cis-platinum, chromate, arsenic trioxide and copper sulfate induced DNA-protein crosslinks at levels that produced acute cytotoxicity, whereas cadmium chloride and lead acetate did not.

Regional Induction of CYP1A1 in Rat Liver Following Treatment with Mixtures of PCB 126 and PCB 153

Liver enzyme induction has been shown previously to be regional with clear borders between induced and uninduced regions in vivo, and cells either fully induced or not induced in vitro. The current study examined this phenomenon in vivo by evaluating enzyme induction after exposure to PCB 126 and PCB 153 in female Fisher 344 (F344) and male Sprague—Dawley (SD) rats. IHC revealed a regional induction of CYP1A1 after exposure to PCB 126, apparent in the centrilobular region at lower doses and progressing to panlobular with higher doses. PCB 153 exposure induced CYP2B1/2 in the centrilobular region, which spread to the midzonal region as the dose increased, but never became panlobular even at the highest dosage tested. In rats treated with PCB 126 in combination with high doses of PCB 153, induction of CYP1A1 occurred preferentially in the periportal region, a reversal from the pattern seen with PCB 126 alone. This CYP1A1 induction pattern reversal is a unique example of complex biological interactions between coplanar (PCB 126) and noncoplanar (PCB 153) halogenated aromatic hydrocarbons.

Polynitroxyl albumin plus tempol attenuates liver injury and inflammation after hepatic ischemia and reperfusion.

PNA+Tempol, albumin containing conjugated (polynitroxyl albumin; PNA) and free (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl; Tempol) nitroxide may protect against injury caused by reactive oxygen species. Therefore, the actions of PNA+Tempol on liver injury and inflammation induced by hepatic ischemia and reperfusion (I/R) were examined. Rats were subjected to 1 h ischemia followed by 24 h reperfusion in the absence (I/R) or presence of PNA+Tempol (25%; 15 mL/kg, i.v.) (I/R+PNA+Tempol) or human serum albumin (23%; 13.5 mL/kg, i.v.) (I/R+HSA). Test solutions were administered prior to and for 2 h during reperfusion. Sham-operated rats underwent surgery with neither ischemia nor infusion. I/R+PNA+Tempol rats had significantly less liver injury and inflammation than I/R rats. I/R+PNA+Tempol livers exhibited focal lesions whereas I/R livers exhibited global necrosis. Likewise, plasma ALT activity was significantly lower in I/R+PNA+Tempol rats. PNA+Tempol reduced I/R-induced neutrophil accumulation and intercellular adhesion molecule-1 (ICAM-1) expression. HSA did not alter I/R-induced liver injury or inflammation. Sham-operated rats exhibited normal liver morphology and no inflammation. Attenuation of I/R liver injury by PNA+Tempol may be mediated by its effect on inflammation, the major contributor to I/R injury. Reduction of inflammation by PNA+Tempol is most likely due to the antioxidative nature of the nitroxides.

Isopropylbiphenyl Metabolism in the Rat. Relationships between Metabolism, Pharmacology and Toxicology

1. 4-Iso[14C]propylbiphenyl was well absorbed following either oral or intraperitoneal administration. Excretion of 14C into faeces was greater than excretion into urine. At 48 h after dosing, appreciable amounts of 14C remained in the rat carcass. 14C was found in tissues examined at 48 h, but was unusually high only in fat.2. Rats with biliary cannulae excreted about half of an oral dose in bile in 48 h.3. The principal metabolite in plasma was biphenylpropionic acid, with small amounts of biphenyl-2-propanol and biphenyl-α-methyl glycolic acid. Unchanged isopropylbiphenyl was also present.4. Metabolites in urine and bile were mainly ring-hydroxylated forms of the plasma metabolites, principally hydroxybiphenylpropionic acid and hydroxybiphenyl-2-propanol. The amount of hydroxybiphenyl-α-methylglycolic acid was unexpectedly small. All phenolic metabolites occurred as conjugates.5. Biphenylpropionic acid is probably the metabolite mainly responsible for the anti-inflammatory action of isopropylbiphenyl, ...

Hepatic inflammatory responses to αα-cross-linked hemoglobin infusion in rats

Abstract Cross-linked hemoglobin (αα-Hb) may be a useful red blood cell substitute if it can be administered safely. However, cell-free hemoglobin has inherent properties that may cause oxidant-mediated toxicity. We investigated whether αα-Hb induces oxidative or inflammatory responses that lead to liver damage. αα-Hb (0.5 or 1.0 gm/kg) was infused into rats, and indices of liver injury, inflammation, and oxidative stress were examined. Although focal hepatic necrosis was noted at 24 hours, plasma alanine aminotransferase activity was not increased and lesions were resolved by 48 hours. Modest neutrophil accumulation in hepatic vessels, but not sinusoids, occurred at 24 hours. Heme oxygenase-1 (HO-1) protein and activity were induced in a dose- and time-dependent manner, with maximal induction at 24 hours. Plasma tumor necrosis factor-α levels were not significantly increased. Additional cytokine- and oxidant-mediated events such as nuclear transcription factor-κB activation and nitric oxide synthase induction were not observed. These results suggest that αα-Hb-derived products such as heme and ferric iron (Fe 3+ ), potent inducers of HO-1, are responsible for increasing HO-1. HO-1 induction may be a protective response by the liver to metabolize excess heme and Fe 3+ , thereby providing antioxidative products to counter the potentially damaging oxidants produced by Fe 3+ -catalyzed reactions.

Liver Toxicity Mediated by Leukocytes and Kupffer Cells

Five presentations comprised this symposium. Leukocyte-mediated drug metabolism was discussed in one talk (J.P.U.). Myeloperoxidase is the primary system responsible for formation of reactive metabolites in neutrophils and monocytes. Metabolites formed directly within leukocytes can inhibit the function of these cells and mediate some of the activities of drugs such as dapsone, propylthiouracil, and 5-aminosalicylic acid. A second presentation (B.H.L.) focused on explaining the low plasma levels of reduced glutathione (GSH) in HIV-infected individuals. Consequently, lymphocyte function is impaired and viral replication is facilitated. The liver is the major source of circulating GSH, and, in AIDS patients, the liver’s capacity to synthesize GSH may be deficient. Data on several in vivo and in vitro models of immunologically mediated liver injury was also discussed (A.W.). The data suggest that, regardless of the initial stimulus and the primary effector cell, a cytokine syndrome with tumor necrosis factor-alpha (TNF) as a major distal mediator is initiated. Cytokine-mediated liver cell injury involves apoptosis. The role of liver macrophages (Kupffer cells) in liver transplantation and alcoholic liver injury was also discussed (R.G.T.). The data implicate cytokines such as TNF and interleukin-6 and deleterious free radicals in the pathophysiology observed in both of these liver disease models. The symposium was concluded with a presentation of data in which the effects of recombinant-DNA derived cytokines, such as TNF, were studied in cultured mouse and rat hepatocytes (R.E.B.). In these cells, both the cytotoxic effects of TNF and the induction of nitric oxide synthase are mediated by oxygen radicals. Glutathione also plays a major role in TNF’s effects. Nitric oxide may function in hepatocytes as a regulator of cytochrome(s) P450.