Walter G. Hanstein

Effects of hexachlorobenzene and iron loading on rat liver mitochondria

The effects of hexachlorobenzene treatment and simultaneous iron-overload on the iron and porphyrin content of rat liver and rat liver mitochondria have been examined. In order to assess damages to the mitochondrial membrane occurring with these treatments, the content of malondialdehyde and selected functional properties of mitochondria were compared with those from control animals. Prolonged intake of hexachlorobenzene (8 weeks) resulted in a strikingly increased level of porphyrins together with a moderate increase in iron concentration. Simultaneous administration of hexachlorobenzene and iron-dextran caused the porphyrin level to reach 25% of the amount induced by hexachlorobenzene alone. The iron concentrations in liver as well as in liver mitochondria are also decreased under these conditions, as compared to the effect of iron-dextran. In contrast, the effects of hexachlorobenzene combined with iron-dextran on mitochondrial oxidative phosphorylation and malondialdehyde content are greater than those of either hexachlorobenzene or iron-dextran. These data suggest that porphyrin accumulation per se causes little deleterious effect and that both agents administered together act synergistically in causing damage to the mitochondrial membrane.

A novel CYP3 gene from female rats

A cDNA library constructed from adult female Sprague Dawley rat liver was screened with polyclonal anti CYP3A-IgG. One of the positive clones, cUT, was found to contain the complete coding sequence of a new gene more similar to hamster gene CYP3A10 (cDNA: 85%, deduced amino acid sequence: 79%) than to the known rat CYP3A genes (cDNA: 75-76%, amino acid sequences: 66-69%). The deduced sequence of the first 28 amino acids is identical to the N-terminus of the testosterone 6 beta-hydroxylase, 6 beta-2 (Nagata, K., Gonzalez, F.J., Yamazoe, Y. and Kato, R. (1990) J. Biochem. 107, 718-725). Northern blots show the presence of cUT mRNA in livers of adult female and male rats. The transcription of the new gene is enhanced in either sex by pregnenolone-alpha-carbonitrile, dexamethasone, phenobarbital, and triacetyloleandomycin, known inducers of CYP3A gene expression.

FORMATION OF LIGAND AND METABOLITE COMPLEXES AS A MEANS FOR SELECTIVE QUANTITATION OF CYTOCHROME P450 ISOZYMES

The suitability of triacetyloleandomycin (TAO) metabolite complex formation and metyrapone binding to reduced cytochrome P450 as a means for selective isozyme quantitation has been studied. Although isozymes of both subfamilies bind metyrapone in the reduced state, selective quantitation of 2B isozymes through the metyrapone complex is possible after complex formation of P450 3A with a TAO metabolite. Thus, consecutive application of both reactions allows the spectroscopic quantitation of P450 3A and 2B isozymes. Complete conversion of P450 3A into the complex, a precondition for P450 3A quantitation, requires NADH in addition to NADPH. A precise collective quantitation of 3A + 2B isozymes as metyrapone complexes alone is not possible because the corresponding complexes possess different molar extinction coefficients, i.e 71.5 and 52 mM-1 cm-1 at 446-490 nm, respectively. The formation of the TAO complex appears to be quite specific, since it correlates well with 3A-specific enzymatic activities, i.e. TAO N-demethylation and formation of 2 beta-hydroxy-, 15 beta-hydroxy- and 6-dehydrotestosterone. P450 3A levels in liver microsomes of male rats either untreated or treated with TAO, dexamethasone (DEX), phenobarbital or hexachlorobenzene amount to 13%, 78%, 66%, 24% and 11% of total P450, respectively. Good correlation between these values and P450 3A-specific enzymatic activities is obtained. By the spectroscopic method, P450 2B isozymes could not be detected in microsomes of untreated rats. With TAO, DEX and hexachlorobenzene the microsomal 2B level is elevated to about 20% of total P450, i.e. to 0.8, 0.4 and 0.4 nmol P450/mg protein, respectively. 2B levels of about 60% of total P450 (0.75 nmol P450/mg protein) are obtained by phenobarbital treatment. Immunoblotting with anti-P450 2B shows that the ratio of expressed 2B1 and 2B2 differs depending on the type of inducer. DEX predominantly leads to induction of 2B2, which may explain the low pentoxyresorufin O-depentylase activity in these microsomes.

Liver microsomal levels of cytochrome P450IA1 as biomarker for exposure and bioavailability of soil-bound polycyclic aromatic hydrocarbons

The bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) for mammalian species was studied with rats fed with a diet containing contaminated soil preparations. The extent of cytochrome P450IA1 (CYP1A1) induction in the liver correlated with the amount of 5- and 6-ring PAHs in the soil samples but not with the total PAH content. Other cytochromes P450 were much less affected by the soil-contaminants. The highest induction of CYP1A1 was obtained with a sample containing 274 mg 5- and 6-ring PAH/kg soil, resulting in a nearly 360-fold increase in the ethoxyresorufin deethylase (EROD) activity. In a semilogarithmic plot, a linear correlation was found between the 5- and 6-ring PAH concentration in the soil and the microsomal CYP1A1 content. As a model for the action of intestinal fluids, soil samples were extracted by bile acid solution. In these experiments, the selectivity in the solubilization of individual PAHs parallels that of toluene extraction, although the yield is lower than the latter and varies with the soil sample. The bioavailability of PAHs for microorganisms, but not for mammals, was shown to be considerably reduced in the presence of high total organic carbon (TOC) values of the soil samples. This may have implications for decontamination strategies, diminishing the effectiveness of biological decontamination in cases with high TOC values. The data suggest that CYP1A1 induction in rats is a parameter that may be useful in risk assessments of contaminated soils for mammalian species.

Energy-dependent accumulation of the uncoupler picrate and proton flux in submitochondrial particles

Abstract In the presence of ATP and oxidizable substrate, submitochondrial particles accumulate up to 7 nmol of picrate/mg of protein. Half of this value is reached at 5 μM picrate in the medium, and maximal energy-dependent accumulation occurs at 25 μM picrate. Mitochondrial proton fluxes calculated under such conditions are 0.80 and 1.08 pmol H + /cm 2 ·sec at 10 μM and 25 μM picrate, respectively. These values are similar to those reported for state 4, and are therefore not large enough for uncoupling by picrate through proton translocation. The energy-dependent spectral response of oxonol VI is reversed to 50 % by 40 μM picrate, suggesting that abolishment of membrane potential is responsible for uncoupling of submitochondrial particles by picrate.

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex

Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-Pi exchange activity. Aurovertin alone inhibits 50% of the oxonol shift at 2 microM, and no further inhibition occurs at up to 12 microM. In combination with even weakly effective uncouplers, 4 microM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10-27%, depending on the preparation. The extent of the oxonol response depends on the ATP/ADP ratio rather than the phosphorylation potential. The dye response in the ATPase complex is 4-7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.

Ökotoxikologische und humantoxikologische Risikobewertung PAK-belasteter Böden vor und nach biologischer Behandlung

ZusammenfassungZiel dieser Arbeit ist es, mögliche toxische Wirkungen PAK-belasteter Böden vor und nach biologischer Sanierung zu erfassen. Hierbei liegt der Schwerpunkt auf der Abschätzung des Risikos für Säugetiere nach oraler Aufnahme von Bodenpartikeln. Als Biomarker-Effekt für die PAK-Aufnahme haben wir in Ratten die Induktion des lebermikrosomalen P450-Enzyms CYP1A1 bestimmt, dessen Expression durch PAK moduliert wird. Die Ergebnisse des Biomarker-Tests wurden mit denen des gleichzeitig eingesetzten Biolumineszenz-Hemmtests (Vibrio fischeri) verglichen, wobei als Testmaterialien jeweils die Original- und die entsprechenden sanierten Bodenproben eingesetzt wurden. Nach biologischer Sanierung PAK-belasteter Böden finden wir je nach Bodenbeschaffenheit und Schadstoffzusammensetzung quantitativ und qualitativ unterschiedliche PAK-Restgehalte in den entsprechenden Bodenproben. Bevorzugt verbleiben die aufgrund ihrer Hydrophobizität schlechter mobilisierbaren höherkernigen PAK in den Materialien. Zusätzlich kann ein hoher organischer Kohlenstoffgehalt der Böden den Schadstoffaustrag in die Wasserphase vermindern und somit hohe Schadstoffrestgehalte nach Sanierung bedingen. Im verwendeten Biolumineszenz-Hemmtest weisen Eluate aller hier untersuchten Bodenproben eine durch Sanierung stark verminderte oder nicht mehr nachweisbare Hemmwirkung auf. Im Gegensatz hierzu hat aber eine biologische Behandlung der Böden kaum einen Effekt auf das Induktionsvermögen für lebermikrosomales CYP1A1 nach oraler Aufnahme durch Ratten. Es zeigt sich, daß die Höhe des Induktionssignals gut mit den Gehalten der Böden an PAK mit 5 und 6 Ringen korreliert, ungeachtet der Tatsache, ob es sich um Original- oder sanierte Böden handelt. Der im Biolumineszenz-Test beobachtete Sanierungserfolg spiegelt sich also im Säugetier-Biomarkertest nicht wider. Hieraus ergeben sich neue, für den Menschen relevante Bewertungskriterien hinsichtlich einer Risikoabschätzung.AbstractThe goal of the present work is to assess the adverse effects of soil bound polycyclic aromatic hydrocarbons (PAH) which remain in soils after biological remediation. We focus on risk assessment for mammalian species with respect to the oral uptake of contaminated soil particles and compare the results of a biomarker test with those of an ecotoxicological assay, the bioluminescence inhibition test withVibrio fischeri. As a biomarker effect in mammals, we determined the liver microsomal cytochrome P450 enzyme CYP1A1 which is induced by PAH in exposed rats. After biological soil treatment, different amounts of PAH remain in the soil depending on the soil properties and initial pollutant composition. Particularly, higher condensated PAH resists biological treatment due to its hydrophobicity. In addition, high amounts of organic carbon in the soils affect remediation efficiency. In the bioluminescence inhibition test, eluates of all biologically treated soils studied do not reveal any or only low inhibitory effects. In contrast, the oral uptake of biologically treated contaminated soils leads to induction levels for CYP1A1 similar to those in the untreated samples. A good correlation is obtained between CYP1A1 levels and the amount of 5 and 6-ring PAH in the soil samples. The main result is that the remediation efficiency determined by the luminescence test is not reflected by the biomarker test, a finding which indicates the high bioavailability of residual PAH in soils. Consequently, new criteria for human risk assessment can be delineated.