Erik Arrhenius

Disturbance of microsomal detoxication mechanisms in liver by chlorophenol pesticides

The pesticide pentachlorophenol known as an uncoupler of mitochondrial oxidative phosphorylation was shown to disturb liver microsomal detoxication functions by a selective inhibition of the terminal oxygenation enzyme P-450. At lower concentrations the flavin moiety of this enzyme chain is not inhibited but rather is stimulated, whereby a qualitative shift in detoxication of aromatic amines from C-oxygenation to N-oxygenation is obtained. The effects were due to the pentachlorophenol itself and not to a metabolite. Similar effects of varying strength were also obtained with other chlorophenol pesticides; 2,4,di-, 2,4,6,-tri and 2,3,4,6-tetrachlorophenol, di- and hexachlorophen, tri- and nonachloro-2-hydroxydiphenyl ethers. The relevance of these findings to the possible synergistic influence of chlorophenols on the carcinogenic effects of polyaromatic amines and hydrocarbons is discussed.

Effects of various in vitro conditions on hepatic microsomal N- and C-oxygenation of aromatic amines.

Abstract The formation of reactive metabolites from carcinogenic aromatic amines in rat liver is associated with microsomal N -oxygenation. C -Oxygenation, i.e. ringhydroxylation and N -dealkylation, yields only harmless metabolites. In an attempt to elucidate the nature and origin of the reactive metabolites, the mechanism of microsomal N - and C -oxygenation of aromatic amines has been studied. Microsomal C -oxygenation of dimethylaniline shows a marked substrate optimum followed by a progressive decline; N -oxygenation of dimethylaniline and N - C -transoxygenation of dimethylaniline- N oxide show a continuous rise with increasing substrate concentration. The inhibition of C -oxygenation at high substrate concentration is not reversed by washing the microsomes. Large amounts of added N -oxygenated metabolite does not affect the C -oxygenation. Detergent treatment decreases C -oxygenation and N - C -transoxygenation but increases N -oxygenation. Electrophilic agents strongly inhibit C -oxygenation but stimulate N -oxygcnation, leaving N - C -transoxygenation unaffected. In the light of these experiments and the electron-donor properties of aromatic amines, the mechanism for C - and N -oxygenation and the release of electrophilic metabolites associated with the latter process is discussed.

Effects of tobacco and tobacco smoke constituents on cell multiplication in vitro

Ascites sarcoma BP8 cells, cultured in suspension in vitro were used as a general toxicity test system for tobacco and tobacco smoke constituents. Some 250 compounds, representative of these materials, were examined by exposing cells to different concentrations of these constituents and measuring the inhibition of culture growth, which was related to corresponding effects encountered for positive standards. When employing the present cell toxicity test system possible effects of factors such as penetration, distribution and microsomal metabolism of the compounds studied, are not taken into account. The most active constituents were found to be unsaturated aldehydes and ketones, phenols and indoles. The good correlation obsered between functional groups and toxicity permits, within the range of functionalities studied, prediction of the toxicity for a compound of known structure.

Subcellular distribution, a factor in risk evaluation of pentachlorophenol.

Pentachlorophenol (PCP) is a potent uncoupler of mitochondrial phosphorylation in vitro and also interferes with microsomal detoxication functions in vitro. This favours flavin mediated oxygenation compared with flavin cytochrome P-450 dependent reactions. Gas chromatographic analysis of subcellular fractions, obtained by zonal centrifugation showed markedly lower PCP concentration in mitochondria and a high accumulation in microsomes compared with cytosol. This increases the likelihood that PCP in vivo causes a malfunction in microsomal detoxication.

Isolated liver perfusion--a tool in mutagenicity testing for the evaluation of carcinogens.

An isolated liver perfusion system suitable for the combination with Chinese hamster V79 cells is described. With this system, it is possible to study, with the V79 cells as genetic targets, the mutagenic effect of a chemical after metabolic activation in the intact organ. Those substances commonly used in mutagenicity testing as inducers of drug metabolising enzymes, i.e. Arochlor 1254. Phenobarbital(PB) and 3-Methylcholantrene(3-MC), were studied for their effect in the isolated perfused liver. PB increased the bile flow, which was not significantly affected by the other inducers. Only Arochlor caused a significant increase in the amino acid incorporation into plasma proteins and total liver proteins (expressed per mg liver protein). None of the inducers had an effect on gluconeogenesis from lactate or urea synthesis. All three inducers caused an increase in the level of microsomal P-450 enzymes, the biggest increase being seen after Arochlor-induction (170%), followed by PB(90%) and 3-MC(50%). Arochlor- and PB-induction had a dramatic effect on N- and C-oxygenation of N, N-dimethylaniline: N-oxygenation was decreased by 35% and 40% respectively and C-oxygenation increased by 130% and 140% respectively. The advantages of the isolated perfused liver as an intact metabolising unit is discussed in relation to other mutagenicity assays, in which subcellular fractions are used as the metabolising system.

Ethanol inhibition of vinyl chloride metabolism in isolated eat hepatocytes

Isolated rat liver cells convert [14C]vinyl chloride into non-volatile metabolites. The metabolism is not increased by in vivo pretreatment with phenobarbital. It is sensitive to inhibition by ethanol, which at a concentration of 4 mM inhibits vinyl chloride metabolism to 50% in hepatocyte suspensions. The metabolic activity is NADPH-dependent and is localized in the microsomal fraction of the liver. The enzyme is also strongly inhibited by tetrahydrofuran, indicating that it could be identical to an ethanol-inducible cytochrome P-450 described in the literature [1].

Dichloro-p-nitroanisole O-demethylase—A convenient assay for microsomal mixed function oxidase in isolated rat hepatocytes

Abstract A new assay for drug metabolism in isolated rat hepatocytes is described. 2,6-Dichloro-4-nitroanisole is O -demethylated to the corresponding phenol. The product, 2,6-dichloro-4-nitrophenol is not further metabolized by the hepatocytes, but accumulates in the extracellular medium. It has a low pK a ; thus the strong colour is pH-insensitive in physiological solutions. The assay involves direct spectrophotometric determination of the product in the medium. The reaction is catalyzed by enzymes present in the microsomal fraction, is dependent on oxygen and NADPH, and it is inhibited by carbon monoxide. The equimolar production of formaldehyde is demonstrated. The results indicate that cytochrome P450 is involved in the reaction.

Dichloro-p-nitroanisole O-demethylase—II. Evidence for separate ethanol inhibited and phenobarbital-inducible enzymes

Abstract Some properties of the monooxygenase activity measured in the recently described dichloro- p -nitroanisole O -demethylase assay were investigated in rat liver microsomes. Enzyme activity in control microsomes was inhibited by ethanol (I 50 = 5 mM). The small slope of the inhibition curve was interpreted as evidence for a heterogenous enzyme system, with at least one enzyme being highly sensitive to ethanol inhibition. A number of other water-miscible organic solvents also inhibited the reaction, tetrahydrofuran being most efficient (I 50 = 0.6 mM). Tween 80 gave only weak inhibition at concentrations up to 4 mg/ml. Vinyl chloride was a strong inhibitor. Phenobarbital pretreatment of the rats increased O -demethylase activity at least 24-fold, and made the enzyme highly sensitive to metyrapone inhibition but not to ethanol. P-448 was not involved in the reaction, since 3-methylcholanthrene pretreatment, or addition of 7, 8-benzoflavone, produced no effect. Only small differences were found between the enzyme from male and female rats.

Some Aspects of Microsomal N- and C-Oxygenation of Aromatic Amines

Since the first demonstration by Mueller and Miller (1949) more than twenty years ago of a NADPH,-dependent microsomal drug metabolizing enzyme, a vast number of foreign and endogenous substances has been shown to be metabolized by this group of microsomal enzymes (Brodie, Gillette & LaDu, 1958). The majority of these reactions also involves molecular oxygen, whereby an oxygenation of the substrate is achieved. The oxygenation may take place at various positions on different substrates. However, this discussion will be limited to the oxygenation of aromatic amines, with certain emphasis on the mechanistic differences and similarities between oxygenation at various carbon atoms (C-oxygenation) on one hand, and oxygenation on the amine nitrogen (N-oxygenation) on the other. The main attention will be given to N-demethylation and N-oxidation of tertiary amines. In the most generally accepted model for the function of the microsomal oxygenating enzyme system, one or several cytochromes (P 450 and the related P 446) are reduced by electrons mediated from NADPH, via a flavin and a possible unidentiSed electron carrier. The reduced cytochrome combines with molecular oxygen which is thereby activated to oxygenate the substrate (Brodie, et al., 1958; This mechanism may involve formation of a ternary cytochrome-oxygen-substrate complex (Hayaishi, 1964). The findings that characteristic spectral changes are induced in liver microsomes by various substrates, even in the absence of NADPH, or dithionite, has led to one major change in this concept, namely, that the substrate may be abIe to combine also with the oxidized forms of P 450 and related cytochromes (Gillette, 1969). The N-oxygenation of aromatic amines however does not fit into this concept. This reaction, which takes place under similar conditions as C-oxygenation is independent of intact P 450 as indicated by the insensitivity of this reaction towards the inhibitory effect of carbon monoxide and disturbances of the intact coupling of flavin and P 450 cytochrome exerted by detergent treatment or ultrasonication (Arrhenius, 1969170 ; Kampffmeyer & Kiese, 1965 ; Ziegler & Pettit, 1964, 1966). Purification of the N-oxygenating enzyme has also given an active preparation virtually free from P 450 (Ziegler, Mitchell & Jollow, 1969). On the basis of these findings Ziegler’s group (Machinist, Orme-Johnson & Ziegler, 1966; Ziegler & Pettit, 1964) and Uehleke (1964) have proposed another model for amine oxygenation involving the formation of N-oxygenated metabolites as intermediates in C-oxygenation. According to this concept the flavoprotein is the oxygenating enzyme, which activates oxygen to oxygenate the

Detoxication disturbances and uncoupling effects in vitro of some chlorinated guaiacols, catechols and benzoquinones

The effects of chlorinated guaiacols, catechols and benzoquinones - earlier identified in discharges from pulp and paper mills -- on oxidative phosphorylation in mitochondria and on detoxication mechanisms in microsomes have been investigated. The same biological systems have been used in testing outgoing water from a sulphite plant. Trichlorocatechol and tetrachlorocatechol and the corresponding guaiacols increase N oxygenation of N,N-dimethylaniline, while C-oxygenation decreases, indicating disturbances in microsomal detoxication. The substances tested have in general an uncoupling effect on mitochondrial oxidative phosphorylation. However, tetrachloroguaiacol seems to differ in having a specific effect on the succinate dehydrogenase part of the respiratory chain. Extracts from waste water from a sulphite plant have a considerable effect on the mitochondrial system such as to produce an increase in basal respiration and a loss of respiratory control. N- and C-oxygenation and phosphorylation have been extensively used in this and other laboratories for evaluating the toxicity of chemicals. The application on water extract described here is rapid and easily handled and thus provides a valuable complement to other water quality tests.