Postmitochondrial Fraction Research Articles

Enzymic reduction of [6]-gingerol, a major pungent principle of ginger, in the cell-free preparation of rat liver

A reductive metabolism of S-(+)-[6]-gingerol [1-(4'-hydroxy-3'-methoxyphenyl)-5-hydroxydecan-3-one], the major pungent principle of ginger, was investigated in vitro with phenobarbital-induced rat liver 10,000 x g supernatant containing the NADPH-generating system. The ethyl acetate-extractable products were isolated and two metabolites were identified as diastereomers of [6]-gingerdiol by gas chromatrography/mass spectrometry. The ratio of two isomers formed in the above reaction was about 1:5, suggesting the stereospecific reduction of S-(+)-[6]-gingerol by carbonyl reductase activity present in the postmitochondrial supernatant fraction of rat liver. The enzymic reduction of S-(+)-[6]-gingerol thus introduces the second asymmetric carbon center in the molecule with concomitant production of S, S- and R, S-isomers of [6]-gingerdiol in different proportions. This stereospecific reduction of [6]-gingerol may be relevant to the clinical use of the compound.

Biochemical and histochemical studies of the effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in mouse brain.

The effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in the mouse brain were studied, and we found that diaphorase activity in the post-mitochondrial fraction of brain homogenate was enhanced by idebenone in a concentration-dependent manner. Histochemical studies also indicated that diaphorase staining was intensified by idebenone at the same concentration. These results suggest that idebenone may stimulate the production of nitric oxide, probably through its direct action on nitric oxide synthase, thus producing its protective action on neurological disorders due to cerebral hypoxia or ischemia as a consequence of dilating the cerebral blood vessels.

Genotoxicity testing with fish hepatic S9 for evaluation of complex mixtures in the aquatic environment: the use of channel catfish as a model

A Salmonella/fish S9 mutagenicity assay, a modification of the traditional rodent Salmonella/musome mutagenicity test of Ames, was evaluated and a working test protocol was established with channel catfish ( Ictalurus punctatus) as a model system for monitoring genotoxins in complex mixtures extracted with organic solvents from freshwater sediments. Post-mitochondrial supernatant fractions (S9) from catfish mediated the biotransformation of the arylamines 2-aminoanthracene (2-AA) and 2-aminofluorene (2-AF) as well as polyaromatic hydrocarbon benzo( a)pyrene (BaP) models to Salmonella (strain TA98) mutagens, confirming other freshwater fish studies with bream, carp, pike, and rainbow trout. The enzymatic activity of catfish S9 was cytochrome P-448-450 like: inducible, heat liable, cofactor dependent, and inhibitor sensitive. Bacterial mutagenesis in the Salmonella/catfish S9 test was dose dependent and sensitive to both S9 concentrations and preincubation activation temperature. A liquid-preincubation modification of the standard plate-incorporation method significantly improved the catfish poikilothermic activation system. The hepatic S9 activity of catfish and the traditional Arochlor-1254-induced rat were qualitatively and quantitatively similar in the detection of model arylamines and benzo( a)pyrene genotoxins. The relative sensitivity of the tests, that is the lowest detectable concentration of 2-AA, 2-AF, and BaP, was <1.0 μg/plate with both activation systems. The sensitivity of the Salmonella/fish S9 assay as a screening tool to detect environmental genotoxins was clearly influenced by three test factors: preinduction of the fish, preincubation of the sample, and optimal preincubation temperature of the fish S9. The successful use of a fish activation system to mediate the biotransformation of progenotoxins establishes this Salmonella/fish S9 mutagenicity test as an ecologically relevant assay to monitor the potential aquatic hazards of environmental genotoxins influencing the aquatic resource of concern - the freshwater fisheries.

Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1

The molecular mechanism of action of vitamin E on mammalian cells remains to be elucidated. In this study, vitamin E dietary intake was assessed for its effects on the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin E dietary intake and aflatoxin B1 (AFB1) genotoxicity measured in vitro. Thus AFB1 induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to effect of vitamin E dietary intake on hepatic microsomal P-450 content and specific activities involved in AFB1 metabolism. Rats were fed ad libitum a diet containing 0, 0.05, 0.5 or 5 IU of α-tocopherol for 8 weeks. Modulation of vitamin E level in postmitochondrial and microsomal fractions resulted in nutritional effects. Cytochrome P-450 content was not modified by the level of vitamin E in the diet. The microsomal P-450 activities, P-450 IIB1 and IIIA, were decreased in the deficient group to −35% and −16%, respectively, as compared with control diet (0.05 IU). Diet supplemented with 0.5 IU of vitamin E increased P-450 IIB and IIIA activities (+28% and +37%, respectively) whereas a diet highly supplemented in vitamin E (5 IU) reduced these specific P-450 activities. Lipid peroxidation, estimated by the formation of thiobarbituric acid reactive products, increased in the dietary vitamin E free diet (+20%) and strongly decreased in the supplemented group (−99%). This study establishes that in vivo, dietary vitamin E protects directly membrane against damage induced by lipid peroxidation and indirectly hepatic microsomal monooxygenase activities. However, vitamin E accumulation seems to alter membrane structure and function. The nutritional effect of vitamin E on hepatic microsomal cytochrome P-450 activities modified the AFB1 genotoxicity measured in vitro.

Pro-oxidant and antioxidant processes in gas gland and other tissues of cod (Gadus morhua)

Partial reduction of molecular oxygen produces reactive oxyradicals, including the superoxide anion radical (O - 2 ) and hydroxyl radical (·OH). The gas gland functions under hyperoxic and acidic conditions and therefore is likely to be subjected to enhanced oxidative stress. Aspects of pro- and antioxidant processes in gas gland were compared with other tissues likely to be subject to differing degrees of oxyradical production, viz. liver (site of chemically-mediated oxyradical production), gills and skeletal muscle. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and total glutathione peroxidase) per g wet weight were highest in liver and lowest in muscle. Catalase and glutathione peroxidase activies per g wet weight were higher in gills than in gas gland, whereas the reverse was seen for superoxide dismutase. Cytosolic superoxide dismutase activities per mg protein were two- and nine-fold higher in gas gland than in liver and gills. The pH characteristics of the antioxidant enzymes were generally similar in all the tissues. Glutathione, vitamin E and unsaturated (peroxidizable) lipid levels were generally highest in liver followed by gas gland. Lipid peroxidation (malonaldehyde equivalents) was evident in all tissues except gas gland. Hydrogen peroxide and O - 2 were involved in the NAD(P)H-dependent ferric/EDTA-mediated formation of ·OH (as measured by 2-keto-4-methiolbutyrate oxidation) by mitochondrial and postmitochondrial fractions of gas gland. Tissue maximal potentials for ·OH production paralled superoxide dismutase but not catalase or glutathione peroxidase activities. Overall, the results confirm the presence of effective antioxidant defences in gas gland and support previous workers' contentions of a central role for superoxide dismutase in this process.

Effect of four plant phenols, β-carotene and α-tocopherol on 3(H)benzopyrene-DNA interaction in vitro in the presence of rat and mouse liver postmitochondrial fraction

The modulatory effect of β-carotene, α-tocopherol and the four plant phenols eugenol, hydroxychavicol, curcumin and catechin on mouse and rat liver postmitochondrial fraction (S9 mix)-mediated 3(H)benzopyrene (B(a)P)-DNA interaction in vitro was studied. All the plant phenolics significantly inhibited 3(H)B(a)P-DNA interaction in the presence of both mouse and rat liver S9 fractions. In contrast, α-tocopherol proved to be ineffective in the presence of both types of S9 mix, whereas β-Carotene inhibited only mouse S9 mix-mediated 3(H)B(a)P-DNA interaction.

Preclinical toxicology studies with azithromycin: genetic toxicology evaluation

Azithromycin was subjected to a series of three in vitro and one in vivo genetic toxicology assays for the detection of drug-associated gene or chromosomal effects. In the Ames Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100, the presence of azithromycin was not associated with any increase in the number of his- revertants. Urine from mice dosed with up to 200 mg/kg of azithromycin also had no effect on the number of revertants in these same strains suggesting the absence of mutagenic excretory products following oral exposure. When tested up to the cytotoxic level of 240 μg/ml, of azithromycin caused to increase in the mutant frequency at the thymidine kinase locus of L5178Y/TK cells. Both the mammalian and microbiol gene mutation assays mutagenic included the presence of rat-liver postmitochondrial (S9) fraction for the detection of mutagenic biotransformation products. Mitogen-stimulated human lymphocytes cultured in the presence of 2.5–7.5 μg/ml azithromycin for 24 h or 30.0–40.0 μg/ml azithromycin for 3 h in the presence of rat S9 had chromosomal aberration frequencies that were no different than negative control cells even though slight to moderate mitotic suppression was associated with these concentrations. In vivo assessment of this compound was completed in male and female mice with a single oral dose of 200 mg/kg followed by sacrifice at 6, 24 or 48 h later and metaphase analysis of bone marrow for chromosomal aberrations. No statistically significant elevations of chromosomally aberrant cells were found. We conclude that azithromycin does not cause gene mutations in microbial or mammalian cells, or chromosomal aberrations in cultured human lymphocytes or in mouse bone marrow in vivo.

Identification and partial characterization of a glycoprotein species with high affinity for methylmercury in peripheral nervous tissues of man and experimental animals.

A small amount of a glycoprotein species (21-kDa glycoprotein) with high affinity for methylmercury (MeHg) was detected in the post-nuclear or post-mitochondrial supernatant fraction of the homogenate of rat sciatic nerve on electrophoresis and autoradiography after binding of Me203Hg to the fraction. The 21-kDa glycoprotein was also found in the subcellular fractions of mouse, hamster, guinea pig, rabbit and human peripheral nervous tissues. Experiments with the cellular fractions of the tissues revealed that the 21-kDa glycoprotein is localized mainly in the myelin fraction, whereas it was not found in the cellular fractions of brain, spinal cord and nonneural tissues, such as kidney and liver. The specific binding activity of the 21-kDa glycoprotein with MeHg was 12-15 fold that of the major myelin protein, Po. It was shown that the interaction of the 21-kDa glycoprotein with MeHg was mediated through sulfhydryl groups in experiments with iodoacetamide and dithiothreitol. The amino acid compositions of the rat and human 21-kDa glycoproteins were similar but very different from that of a typical metallothionein. The N-terminal amino acid sequences of the two components of the rat 21-kDa glycoprotein were identical to those of P0 and PMP-22, respectively. The in vitro binding of MeHg was also observed in the myelin fraction obtained from the sciatic nerves of MeHg-dosed rats.

Activation of benzo[ a]pyrene and 2-aminoanthracene to bacteria mutagens by mussel digestive gland postmitochondrial fraction

The ability of the mussel postmitochondrial fraction (S9) to activate benzo[ a]pyrene (BaP) and 2-aminoanthracene (2AA) to mutagenic metabolites towards Salmonella typhimurium strain TA98 was tested. The mechanisms involved in this activation were investigated and mussel cytochrome P-450-dependent monooxygenases and its NADPH cytochrome c reductase were found to contribute to the activation of BaP. This activation was improved by treating the mussel with 4,5,4′,5′-tetrachlorobiphenyl (TCB) (a 3-methylcholanthrene-type inducer of cytochrome P-450-dependent monooxygenase in marine fish) and was inhibited by α-naphthoflavone (ANF), a cytochrome P-450 inhibitor. However, both BaP activation and cytchrome P-450-related metabolic activities are much weaker in mussels than in vertebrates. Mussel S9 activates aromatic amines more effectively than BaP. Pretreatment of mussels with TCB or addition of ANF in the incubation medium has no effect on 2AA activation. As suggested by Kurelec (1985), aromatic amine metabolism may be supported by a flavoprotein mixed-function amine oxidase which is NADPH-dependent.

Effects of fasting on phosphatidylcholine biosynthesis in hamster liver: regulation of cholinephosphotransferase activity by endogenous argininosuccinate.

The control of phosphatidylcholine biosynthesis in the hamster liver was examined. Livers of hamsters fasted for 24 and 48 h were perfused with labelled choline. Under both fasting conditions, the incorporation of labelled choline into phosphatidylcholine was reduced. After 48 h of fasting, the 52% reduction in phosphatidylcholine biosynthesis was caused by changes in several factors including a diminishing rate of choline uptake and severe reductions in the pool sizes of ATP and CTP (to 33-37% control values) which resulted in a decrease in the pools of choline-containing metabolites. The activation of cytidylyltransferase after 48 h of fasting might be regarded as a compensatory mechanism for the maintenance of phosphatidylcholine biosynthesis. After 24 h of fasting, a 25% reduction in phosphatidylcholine biosynthesis was observed. The ATP and CTP levels were decreased but the reduction was not severe enough to affect the choline uptake or the labelling of the phosphocholine fraction. The activities of the cytidylyltransferase remained unchanged but an accumulation of labelled CDP-choline was detected. Although choline-phosphotransferase activity was not changed in the microsomes, the enzyme activity was attenuated in the postmitochondrial fraction. Further analysis revealed that cholinephosphotransferase in the liver was inhibited by an endogenous inhibitor in the cytosol which was later identified as argininosuccinate. The level of argininosuccinate was elevated during fasting and the change quantitatively accounted for the attenuation of cholinephosphotransferase activity. The inhibition of choline-phosphotransferase by argininosuccinate, coupled with a substantial decrease in the diacylglycerol level, would provide the hamster liver with an immediate mechanism for the transient modulation of phosphatidylcholine biosynthesis during short-term fasting.

Fate of fluazifop butyl in rat and human skin in vitro.

Enzyme mediated hydrolysis of fluazifop butyl has been measured with rat and human skin post-mitochondrial fractions. Rat skin had a ten times greater capacity to metabolise fluazifop butyl than human skin, but the enzyme affinities were similar. The post-mitochondrial fraction metabolism was compared to that seen during absorption in a flow through diffusion cell with viable skin. Limited hydrolysis of absorbed fluazifop butyl was seen in rat skin, but increased two fold if the stratum corneum was removed. The stratum corneum was found to retain fluazifop butyl. When the skin was pre-incubated with the esterase inhibitor bis (p-nitrophenol) phosphate (BNPP), reduced metabolism was seen. No metabolism of fluazifop butyl was seen in human skin during absorption. Retention of the compound by the stratum corneum is postulated to restrict the accessibility of the compound to the enzyme site, thus influencing the observed metabolism during the absorption process.

Influence of Starvation and Chronic malnutrition on Lipid Peroxidation in Rats Treated with Ethanol.

The effect of starvation and chronic food restriction on lipid peroxidation during treatment with ethanol was studied. The experiment was carried out on 64 female Wistar rats. The formation of malondialdehyde was determined in postmitochondrial liver fraction (basal level and after stimulation with ascorbate). The concentration of reduced glutathione was measured in the same fraction.In rats fed ad libitum, the consumption of ethanol, given with the drinking water for either 3 or 28 days, promoted a slight, but statistically nonsignificant, activation of lipid peroxidation. In animals not given ethanol, a 3-day fast increased the formation of lipid peroxides after stimulation with ascorbate. However, some tendency toward inhibition of lipid peroxidation was established after consumption of a restricted diet (6-8g standard pellet food daily) for 28 days. The treatments with alcohol for 3 and 28 days were combined respectively with a 3-day fast and a 28-day food restriction. In both a statistically significant increase in lipid peroxides was observed in comparison with the controls, which were fed ad libitum and were not given ethanol.The concentration of reduced glutathione was decreased in all experimental groups, but only with the 3-day treatments did the extent of its depletion correspond to the activation of lipid peroxidation. The results show that both starvation and chronic malnutrition potentiate lipid peroxide formation in rats treated with ethanol.

Sulfotransferase-mediated mutagenicity of 1-hydroxymethylpyrene and 4H-cyclopenta[def]chrysen-4-ol and its enhancement by chloride anions.

1-Hydroxymethylpyrene (HMP), a primary benzylic alcohol, and 4H-cyclopenta[def]chrysen-4-ol (OH-CPC), a secondary benzylic alcohol, were investigated for mutagenicity in Salmonella typhimurium (reversion of the his- strain TA98) in the presence of various xenobiotic-metabolizing systems. In the direct test, HMP was inactive and OH-CPC was very weakly active. In the presence of NADPH-fortified postmitochondrial fraction from rat liver (S9/NADPH), no activation of OH-CPC was observed, whereas strong mutagenic effects were elicited by HMP. In the presence of cytosol and 3'-phosphoadenosine-5'-phosphosulfate (PAPS), both alcohols were activated to potent mutagens. For equal mutagenic effects, approximately 650-fold lower concentrations of HMP were required in the cytosol/PAPS-mediated assay than in the S9/NADPH-mediated assay. The cytosol/PAPS-mediated mutagenicity of both alcohols was 3- to 4-fold enhanced, when KCl (125 mM) was present during the exposure. The authentic chloromethylarenes, 1-chloromethylpyrene and 4-chloro-4H-cyclopenta[def]chrysene, showed very strong direct mutagenicity. These results, taken together with previous findings, indicate that both primary and secondary benzylic alcohols derived from polycyclic aromatic hydrocarbons may be activated by sulfotransferases to electrophilic sulfuric acid esters, and by subsequent substitution reaction to further active species such as benzylic chlorides.

Clavine alkaloids and derivatives as mutagens detected in the Ames test.

Eight cytostatic clavines were investigated for mutagenicity in Salmonella typhimurium (reversion of the his-strains TA98, TA100, TA102 and TA1537), directly and in the presence of a mammalian xenobiotic metabolizing system, S9 (NADPH-fortified postmitochondrial fraction of liver homogenate from Aroclor 1254-treated rats). Four compounds (festuclavine, 17-bromofestuclavine, 1-allylelymoclavine and 1-methyllysergol methyl ether) were direct mutagens, whose activity was enhanced in the presence of S9. The other compounds (1-cyclopentylfestuclavine, 13-bromo-1-cyclopropylmethylfestuclavine, 6-cyano-1-propyl-6-norfestuclavine and 6-allyl-1-propyl-6-norfestuclavine) showed mutagenic effects only in the presence of S9, as previously observed with other clavines (agroclavine and its 1-propyl and 1-pentyl derivatives). Thus, all investigated clavines may be metabolized to mutagenic products by mammalian enzymes. Bacteriotoxic activities did not correlate with mutagenic activities. The bacteriotoxicity of several clavines was reduced in the presence of S9. The results are discussed with regard to the potential therapeutic use of clavine alkaloids as antimicrobial and antineoplastic agents.

Passive avoidance learning in the young chick results in time- and locus-specific elevations of alpha-tubulin immunoreactivity.

A monoclonal antibody was used to examine changes in immunoreactivity of the cytoskeletal protein, alpha-tubulin, following passive avoidance learning in day-old chicks. Postmitochondrial fractions (16,000 g supernatants) were prepared from specific forebrain loci taken at several time points after training and assayed with the anti-alpha-tubulin antibody, YL1/2. Of the regions examined, elevations in the titre of YL1/2 were found in the left intermediate hyperstriatum ventrale 1 h, 6 h and 24 h following training, in the left lobus parolfactorius 1 h following training and in the right lobus parolfactorius 6 h and 24 h following training. No training-related changes were detected in a third forebrain region, the paleostriatum augmentatum. These results regarding the cellular dynamics of memory formation in the chick confirm and expand on earlier findings from our laboratory.

Effect in vitro of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the hepatic activation of dietary genotoxins by rat post-mitochondrial fractions

The effect of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the conversion of the heterocyclic amine 2-amino-3-methylimidazo[4,5-ƒ]quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay. The assay used Salmonella typhimurium TA98 as an indicator of the mutagenicity and hepatic post-mitochondrial fractions (S-9) from male Sprague-Dawley rats as the activating system. All three fatty acids inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA repair processes within the bacterial cell. The activation of three other food mutagens, 2-amino-3,8-dimethylimidazo[4,5-ƒ]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and aflatoxin B 1 (AFB 1) was also inhibited by these fatty acids.

Mitochondrial oxidative stress after carbon monoxide hypoxia in the rat brain.

To better understand the mechanisms of tissue injury during and after carbon monoxide (CO) hypoxia, we studied the generation of partially reduced oxygen species (PROS) in the brains of rats subjected to 1% CO for 30 min, and then reoxygenated on air for 0-180 min. By determining H2O2-dependent inactivation of catalase in the presence of 3-amino-1,2,4-triazole (ATZ), we found increased H2O2 production in the forebrain after reoxygenation. The localization of catalase to brain microperoxisomes indicated an intracellular site of H2O2 production; subsequent studies of forebrain mitochondria isolated during and after CO hypoxia implicated nearby mitochondria as the source of H2O2. In the mitochondria, two periods of PROS production were indicated by decreases in the ratio of reduced to oxidized glutathione (GSH/GSSG). These periods of oxidative stress occurred immediately after CO exposure and 120 min after reoxygenation, as indicated by 50 and 43% decreases in GSH/GSSG, respectively. The glutathione depletion data were supported by studies of hydroxyl radical generation using a salicylate probe. The salicylate hydroxylation products, 2,3 and 2,5-dihydroxybenzoic acid (DHBA), were detected in mitochondria from CO exposed rats in significantly increased amounts during the same time intervals as decreases in GSH/GSSG. The DHBA products were increased 3.4-fold immediately after CO exposure, and threefold after 120 min reoxygenation. Because these indications of oxidative stress were not prominent in the postmitochondrial fraction, we propose that PROS generated in the brain after CO hypoxia originate primarily from mitochondria. These PROS may contribute to CO-mediated neuronal damage during reoxygenation after severe CO intoxication.

Pesticides as activators of mouse liver microsomal glutathione S-transferase

Selected sulfhydryl-reactive pesticides activate mouse liver microsomal glutathione S-transferase (GST m) assayed in purified form free from cytosolic transferases (GST cs) with 1-chloro-2,4-dinitrobenzene as the substrate. Maximum activations were as follows: 1315% for N-ethylmaleimide (positive control) at 1 m M, 374% for chloranil at 0.01 m M, 272% for EPTC sulfoxide at 10 m M, 255% for captan at 0.1 m M, 228% for acrolein at 1 m M, and 152% for methyl isothiocyanate at 10 m M. Pesticides which showed little or no activation were alachlor, atrazine, tetramethrin, and tridiphane. The toxicological relevance of GST m activation was examined with the aforementioned activators plus EPTC and allyl alcohol. Experiments were conducted in vitro or in vivo with washed microsomes (which contain not only GST m but also some GST cs) and the post-mitochondrial supernatant fraction with its high GST c activity. These studies established that GST m, either unactivated or activated, was not a significant contributor to overall mouse liver GST activity.

On metallothionein, cadmium, copper and zinc relationships in the liver and kidney of adult rats.

1. A short-term exposure of adult Wistar rats to Cu (50 micrograms/ml) and Cd (10.0 micrograms/ml drinking water) caused significant changes in the subcellular concentrations of Cd, Cu, Zn and metallothionein (MT) in the liver and kidney; the concentrations were close to the physiological values, however. 2. To establish a relationship between these changes in the subcellular concentrations of Cd, Cu, Zn and the level of MT in the post-mitochondrial fraction of the liver and kidney, the analytical data (N = 42) were subjected to the multiple regression analysis. 3. The analysis showed that MT synthesis in the liver was principally induced by small amounts of Cd (0.32-1.4 micrograms/g wet wt) whereas in the kidney a level of MT in the post-mitochondrial fraction correlated positively with the renal Cd and Cu, as well as with the level of this protein in the liver. 4. The above results together with the positive correlation between the level of MT in the post-mitochondrial fraction and the concentration of Cu in this fraction, as well as the fact that under normal physiological conditions the capacity of MT (beta-domain) in the liver and kidney was sufficient to bind 50-100% of the total post-mitochondrial Cu suggest that MT, first induced by small amounts of Cd, may be involved in the metabolism of Cu.

Comparative affinity of steroidal and non-steroidal antioestrogens, cholesterol derivatives and compounds with a dialkylamino side chain for the rat liver antioestrogen binding site

Steroidal and non-steroidal antioestrogens, steroidal compounds with (disubstituted) dialkyl amino side chain, cholesterol derivatives, histaminic and (anti)-progestational compounds were tested for their ability to compete with [ 3H]tamoxifen for the specific antioestrogen binding site (AEBS) in the post-mitochondrial fraction of rat liver homogenates. Relative binding affinity was highest for compounds with diethylamino or pyrrolidino ethoxy side chains. Affinity decreased with shortening of this side chain. No connection could be established between the carbon backbone of the compound and affinity, except for the presence of (sometimes aromatic) ring structures. Steroidal ring structures do not seem to be necessary for binding. the cholesterol derivatives, tested in our laboratory, showed very little affinity for the rat liver AEBS. Histamine, melatonin and the (anti)-progestational compounds showed no affinity for the AEBS; and we therefore conclude that the AEBS is not identical to receptors for these compounds. Results of these experiments could be useful in other investigations on the development of resistance of breast cancer to antioestrogens.