Douglas E. Goeger

Porphyria cutanea tarda: multiplicity of risk factors including HFE mutations, hepatitis C, and inherited uroporphyrinogen decarboxylase deficiency.

The coexistence of factors considered to contribute to development of porphyria cutanea tarda was studied in 39 consecutive patients. Highly prevalent factors were alcohol intake in 79%, smoking in 86%, hepatitis C virus infection in 74%, estrogen use in 73% of 11 females, and at least one mutation in the HFE (hereditary hemochromatosis) gene in 65%. The C282Y mutation was found in 29%, H63D in 47%, and S65C in 0%. HFE genotypes included C282Y/C282Y in 9%, H63D/H63D in 9%, C282Y/H63D in 12%, C282Y/wild type in 9%, and H63D/wild type in 26%. Less prevalent were HIV infection in 15% (or 25% of those tested, N = 24) and erythrocyte uroporphyrinogen decarboxylase deficiency, which distinguishes familial (type 2) from “sporadic” (type 1) porphyria cutanea tarda, in 19%. Multiple contributing factors coexisted in both types 1 and 2, with 92% of all patients having three or more factors. These observations indicate that this porphyria is multifactorial in the individual patient, and therefore is seldom attributable to a single identifiable cause. Profiling for all potentially contributing factors is important for individualizing management.

Comparison of three tetramic acids and their ability to alter membrane function in cultured skeletal muscle cells and sarcoplasmic reticulum vesicles

Cyclopiazonic acid is a potent inhibitor of calcium uptake and Ca(2+)-ATPase activity in sarcoplasmic and endoplasmic reticulum. In L6 muscle myoblasts, cyclopiazonic acid stimulates the uptake of tetraphenylphosphonium, a lipophilic membrane potential probe, and has antioxidant properties. The purpose of the present study was to investigate the structural requirements necessary for causing the surface charge alterations, and the antioxidant activity in L6 skeletal muscle myoblasts, and for inhibition of calcium transport by rat skeletal muscle sarcoplasmic reticulum vesicles. This was accomplished by comparing the effects of two structurally related tetramic acids, cyclopiazonic acid imine and tenuazonic acid, with cyclopiazonic acid. Cyclopiazonic acid imine inhibited oxalate-assisted 45Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles and stimulated tetraphenylphosphonium accumulation by L6 muscle myoblasts. However, these effects required an approximately fourfold higher concentration than that of cyclopiazonic acid. Tenuazonic acid, up to 1 mM, had no effect on oxalate-assisted 45Ca2+ uptake or Ca(2+)-ATPase activity in sarcoplasmic reticulum vesicles and did not stimulate tetraphenylphosphonium accumulation by L6 muscle myoblasts. Cyclopiazonic acid was only slightly more effective than cyclopiazonic acid imine at preventing the patulin-induced increase in thiobarbituric acid positive substance (used to estimate lipid peroxidation); tenuazonic acid was totally ineffective. Previously, it was shown that cyclopiazonic acid was twice as effective as cyclopiazonic acid imine at preventing increases in thiobarbituric acid positive substance in cultured renal cells, LLC-PK1. Thus, the indole nucleus of cyclopiazonic acid is essential for the membrane-associated biological activity; however, modification of the acetyl group reduces the potency of the activity.

Interference of 6β-Hydroxycortisol in the Quantitation of Urinary Free Cortisol by Immunoassay and Its Elimination by Solid Phase Extraction

Abstract Objectives: To study the cross-reactivity of 6β-hydroxycortisol (6β-OHF) with anticortisol antibodies and subsequent interference with urinary free cortisol (UFC) quantitation in commercial immunoassay kits. A solid-phase-extraction (SPE) technique was evaluated for removal of 6β-OHF from the specimen. Methods: Interference by 6β-OHF was studied in three enzyme immunoassay and three radioimmunoassay kits. Interference was assessed by the multiple regression equation derived from a 4 × 4 matrix composed of four levels of cortisol and four levels of 6β-OHF. Sep-Pak Plus C18 cartridges were used to remove 6β-OHF from the specimens, and optimum conditions for fractionating 6β-OHF from cortisol were determined by eluting the cartridge with a stepwise increment in methanol concentration. HPLC was used to monitor cortisol and 6β-OHF in the sample and cartridge eluates. Results: Cross-reactivity of 6β-OHF with anticortisol antibodies was variable and depended on the cortisol kit used with a range from 0.1 to 10%. Multiple regression analysis indicated that 6β-OHF interfered positively with the cortisol assay regardless of cortisol concentrations. Extraction of UFC by methylene chloride as recommended by some immunoassay kits yielded a significant constant error to the UFC results which was nearly proportional to the added levels of 6β-OHF. SPE of samples completely removed 6β-OHF from the specimen when a 40% methanol elution step was included. Consequently, specimens processed in this manner did not show interference in UFC measurements even in samples spiked with 2.38 μmol/L of 6β-OHF. Conclusions: Elevated urinary 6β-OHF may be a significant source of interference in UFC immunoassays. SPE of samples prior to analysis would be a simple and inexpensive means for removing 6β-OHF from urine specimens, therefore, increasing accuracy and precision in UFC measurements by immunoassay.

Co-mutagenicity of Coumarin (1,2-benzopyrone) with Aflatoxin B1 and Human Liver S9 in Mammalian Cells

Coumarin (1,2-benzopyrone), a natural dietary constituent and drug currently under evaluation for treatment of certain cancers and lymphedema, reduces polycyclic aromatic hydrocarbon-induced neoplasms in rodents. Because most rodents metabolize coumarin through 3,4-epoxidation, whereas 7-hydroxylation predominates in humans, their suitability as a model for coumarin effects in humans has been questioned. We examined coumarin chemoprotection against the promutagen and dietary contaminant aflatoxin B1 with human liver S9 bioactivation in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase mutation assay. Coumarin in the absence of aflatoxin B1 was not mutagenic or cytotoxic up to 500 microM. When included with either 1 or 10 microM aflatoxin B1, coumarin produced a dose-dependent increase in mutant frequency and cytotoxicity. At concentrations greater than 50 microM, coumarin stimulated human liver S9 bioactivation of aflatoxin B1 to the mutagenic 8,9-epoxide. This increase was 12- and fivefold at 500 microM coumarin with 1 and 10 microM aflatoxin B1, respectively, compared with incubations with aflatoxin B1 alone. These findings differ from previous results with liver S9 from other species, and indicate that coumarin co-mutagenicity with aflatoxin B1 and human liver S9 is through increased aflatoxin B1 bioactivation.

Effects of chloroquine in hematoporphyrin-treated animals

Porphyrins and related compounds are useful in photodynamic therapy but can cause cutaneous photosensitivity. We determined whether chloroquine, which is effective in treating porphyria cutanea tarda, would mobilize an administered porphyrin from tissues and enhance its excretion. Hematoporphyrin with and without chloroquine was administered to chick embryos, mice, and rats. Tissue and plasma porphyrin levels were markedly increased after hematoporphyrin dosing. Porphyrin concentrations in liver, spleen, and kidney were not significantly affected by chloroquine. Total urinary and fecal porphyrin excretion in rats treated with hematoporphyrin (50 mg/kg, i.p.) was not influenced by chloroquine treatment (100 mg/kg, s.c.). Excretion of heptacarboxylporphyrin, normally a minor fraction of urinary porphyrins, was significantly increased in chloroquine-treated rats. These results suggest that chloroquine is unlikely to be useful after photodynamic therapy for mobilizing exogenous porphyrins from tissues such as liver, spleen, and kidney. Increased urinary excretion of heptacarboxylporphyrin may contribute to the beneficial effect of chloroquine in porphyria cutanea tarda.

Coumarin chemoprotection against aflatoxin B1-induced gene mutation in a mammalian cell system: A species difference in mutagen activation and protection with chick embryo and rat liver S9

Coumarin (1,2‐benzopyrone), a natural food constituent, prevents polycyclic aromatic hydrocarbon‐induced neoplasms in rats and mice, but has not been studied with other chemical carcinogens. We examined coumarin chemoprotection against aflatoxin B1 using the 6‐thioguanine resistance mutation assay in two different Chinese hamster ovary cell lines (K1BH4 and AS52) with liver S9 from rats and 19‐day‐old chick embryos for aflatoxin B1 bioactivation. Laboratory rodents metabolize coumarin through 3‐hydroxylation, whereas 7‐hydroxylation predominates in chick embryos and humans. Chick embryo liver S9 was approximately 25‐fold more effective in activating aflatoxin B1 to the mutagenic and cytotoxic metabolite(s) than rat liver S9. Coumarin added at 50 and 500 μM with chick embryo liver S9 reduced the mutant frequency of 1 μM aflatoxin B1 by 40 and 85%, respectively. Coumarin up to 500 μM had no effect on aflatoxin B1 mutagenicity with rat liver S9. When liver S9 from chick embryos pretreated with coumarin was used for aflatoxin B1 bioactivation, mutant frequency and cytotoxicity were decreased compared to liver S9 from vehicle‐treated controls. Liver S9 from coumarin‐treated rats did not significantly affect mutant frequency or cytotoxicity. HPLC analysis of chick embryo liver S9 incubated with 1 μM aflatoxin B1 showed a dose‐dependent decrease by coumarin of aflatoxin B1 activation to the 8,9‐epoxide ranging from 70% of controls at 5 μM coumarin to 4% of controls at 500 μM coumarin. In contrast, coumarin produced a dose‐dependent increase in 20 μM aflatoxin B1 activation by rat liver S9, reaching twice the control levels at 500 μM coumarin. These findings, using a mammalian cell system as a mutagenic endpoint, demonstrate marked species differences in chemoprotection by coumarin. Environ. Mol. Mutagen. 32:64–74, 1998

Biochemical characterization of coumarin 7-hydroxylase activity in chick embryo liver microsomes

Coumarin occurs naturally in the diet and can induce and inhibit cytochrome P450 enzymes. Hepatic coumarin 7-hydroxylase activity is the major pathway for coumarin metabolism in humans but not in rats, most strains of mice, or other laboratory animals. Coumarin 7-hydroxylase activity and the effects of chemical inhibitors and inducers on this activity were studied in 19-day-old chick embryo liver microsomes. Activity was between 35 and 75 nmol/mg protein/hr which is approximately 2-fold higher than reported for human liver microsomes. The pH optimum was 7.8 and the Km determined by both an ether extraction and a high performance liquid chromatography method was 7.3 +/- 0.9 (+/- SD) microM. Substrate inhibition was evident at coumarin concentrations above 250 microM (activities at 1000 and 4000 microM coumarin were 84 and 40% of Vmax, respectively). The Ki values (+/- SD) for inhibitors of microsomal coumarin 7-hydroxylase activity in vitro were: alpha-naphthoflavone, 46.9 +/- 19.8 nM; metyrapone, 0.8 +/- 0.9 microM; aniline, 12.3 +/- 8.2 microM; cimetidine, 70.9 +/- 27.9 microM; N-nitrosodimethylamine, 0.7 +/- 0.9 mM; and dimethyl sulfoxide, 7.9 +/- 1.9 mM. Treatment of chick embryos with pyrazole (40 mumol) increased coumarin 7-hydroxylase by 50% at 24 hr, but this activity was unaffected by treatment of embryos with 3-methylcholanthrene (2 mumol) or glutethimide (8 mumol). Thus, hepatic coumarin 7-hydroxylase activity in 19-day-old chick embryos is higher than in most laboratory animals and has similar biochemical properties as the enzyme in humans and mice. The chick embryo liver may be a useful system for studies on the biochemical effects of coumarin and the regulation of cytochrome P450-dependent coumarin 7-hydroxylase.