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C Ioannides

Publications and source records attributed to C Ioannides.

At least 19 recordsLinked to original sources
PubMed2001

A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.

Androstenedione↗
PubMed2001

Mutagenicity of bay-region amino-substituted cyclopenta[a]phenanthrenes and 2- and 5-aminochrysene.

The relative mutagenic potentials of 11-amino-16,17-dihydro-15H-cyclopenta[a]phenanthrene, its 17-keto derivative, and 2- and 5-aminochrysene have been compared in Salmonella typhimurium TA98 and TA100 in the presence of a postmitochondrial liver preparation from Aroclor 1254 induced rats. The 11-amino hydrocarbon is a very weak mutagen (0.27 revertants/nmol), whereas the 11-amino-17-ketone is much more active (129 revertants/nmol). 2-Aminochrysene is the most mutagenic arylamine ( approximately 500 revertants/nmol) among these compounds, but its 5-amino isomer is much less active (0.9 revertants/nmol). Possible reasons for these marked differences are suggested. Use of TA98 with over-expressing O-acetyltransferase (YG 1024) and deficient in this enzyme (TA98/l,8-DNP(6)) with the 11-amino-17-ketone and with 5-aminochrysene clearly indicates the importance of this enzyme in their bioactivation, implying oxidation of the amino group to the hydroxylamine in both these compounds.

Androstenes↗
PubMed2001

The metabolism and bioactivation of agaritine and of other mushroom hydrazines by whole mushroom homogenate and by mushroom tyrosinase.

Whole homogenates of Agaricus bisporus metabolised the mushroom hydrazine agaritine [beta-N-(gamma-L(+)glutamyl)-4-(hydroxymethyl) phenylhydrazine] to generate at least three metabolites. None of these metabolites, however, was the free hydrazine [4-(hydroxymethyl)phenylhydrazine], the postulated metabolite of agaritine believed to be formed as a result of the loss of the gamma-glutamyl group, the reaction being catalysed by gamma-glutamyltransferase. The three metabolites of agaritine displayed weak mutagenic activity towards Salmonella typhimurium strain TA104. 4-(Hydroxymethyl)phenylhydrazine, as the N'-acetyl derivative, was metabolised by mushroom tyrosinase to yield a number of metabolites that induced a mutagenic response in S. typhimurium TA104. Similar to N'-acetyl-4-(hydroxymethyl)phenylhydrazine, agaritine was extensively metabolised by the mushroom tyrosinase but, in contrast, the structurally related N'-acetyl-4-hydrazinobenzoic acid did not serve as substrate of this enzyme, implying a critical role for the hydroxymethyl group at the para-position. In conclusion, the current studies have demonstrated for the first time that: (a) whole mushroom homogenates readily metabolise agaritine but not to the postulated 4-(hydroxymethyl)phenylhydrazine; and (b) mushroom tyrosinase metabolises agaritine and N'-acetyl-4-(hydroxymethyl)phenylhydrazine, in the latter case forming genotoxic metabolites.

Agaricales↗
PubMed2001

Contribution of CYP1A2 in the hepatic metabolism of melatonin: studies with isolated microsomal preparations and liver slices.

The objective of the present studies was to define the enzyme systems catalysing the 6-hydroxylation of melatonin, by monitoring the levels of 6-sulphatoxymelatonin in rat hepatic postmitochondrial preparations and in precision-cut liver slices. Melatonin 6-hydroxylase activity was localized in microsomes and was supported by NADPH, but not NADH. Treatment of rats with beta-naphthoflavone more than tripled 6-sulphatoxymelatonin formation from melatonin, but gave rise only to a moderate increase (25%) in the sulphate conjugation of 6-hydroxymelatonin. Treatment of rats with phenobarbitone, acetone, dexamethasone and clofibrate did not increase 6-sulphatoxymelatonin generation when either melatonin or 6-hydroxymelatonin served as substrates. Of a number of cytochrome P450 inhibitors investigated, only furafylline inhibited markedly the conversion of melatonin to 6-sulphatoxymelatonin without any concomitant effect on the sulphoconjugation of 6-hydroxymelatonin. When liver slices were incubated with melatonin, treatment of rats with beta-naphthoflavone, and to a lesser extent phenobarbitone, elevated the levels of 6-sulphatoxymelatonin in the culture medium. No such increase was seen when slices from beta-naphthoflavone-treated rats were incubated with 6-hydroxymelatonin, whereas a modest increase was seen with slices from phenobarbitone-treated rats. Treatment of rats with acetone, dexamethasone or clofibrate failed to modulate the levels of 6-sulphatoxymelatonin generated from either melatonin or 6-hydroxymelatonin. Molecular modelling analysis revealed that melatonin had a high area/depth(2) ratio, displayed characteristics of CYP1A2 substrates and could be readily accommodated into the human CYP1A2 active site in a position favouring 6-hydroxylation. Collectively, all the above data provide strong experimental evidence that CYP1A2 is an important catalyst of the 6-hydroxylation of melatonin.

Animals↗
PubMed2001

Effect of antioxidant flavonoids and a food mutagen on lymphocytes of a thalassemia patient without chelation therapy in the Comet assay.

Thalassemia remains a significant health problem in Europe, the Middle East, and Asia. In such patients, generally high iron levels make free oxygen radicals accessible, for example, through Fenton-type chemistry, and generate superoxide and hydroxyl radicals. Increased oxygen radical capacity is known to be associated with cancer and ageing. It was shown in previous studies that peripheral blood lymphocytes from a sickle/beta thal double heterozygote-sickle phenotype, thalassemia patient, not yet on chelation therapy, were more sensitive to the effects of oxygen radicals and iron salts than lymphocytes from normal controls. Iron overload in thalassemia patients can result from dietary absorption. It was considered that with other dietary agents, such as food mutagens and flavonoids, the thalassemia patient might also show increased sensitivity to the effects of these agents. The present study, therefore, compared the effects of the food mutagen/carcinogen, 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2), in fresh or frozen normal human peripheral lymphocytes with frozen lymphocytes from the same thalassemia patient. The lymphocytes from the thalassemia patient showed an approximately two-fold increase in sensitivity. When a combination of Tryp-P-2, with either quercitin or kaempferol, was compared in frozen lymphocytes and lymphocytes from the thalassemia patient, a two-fold increase in sensitivity was also maintained. Responses to Trp-P-2 were reduced to untreated control levels at the highest doses of quercitin and kaempferol, and were highly significantly different by comparison with Trp-P-2 alone (P<0.001). The flavonoids acted in an antigenotoxic/antioxidant manner. Sensitivity was slightly increased with kaempferol by comparison with quercitin. At low concentrations of the flavonoids there was some evidence of an exacerbation of response, perhaps due to a switch to pro-oxidant status. This exacerbation of response at low doses of flavonoids has been seen in earlier studies with normal lymphocytes. Teratogenesis Carcinog. Mutagen. 21:165-174, 2001.

Carbolines↗
PubMed2000

Quantitative structure-activity relationships in a series of endogenous and synthetic steroids exhibiting induction of CYP3A activity and hepatomegaly associated with increased DNA synthesis.

The results of a quantitative structure-activity relationship (QSAR) study on a total of 14 steroids exhibiting induction of a CYP3A-associated activity and increase in liver weight/DNA synthesis is reported. It is found that different, but related, structural descriptors correlate with increase in ethylmorphine N-demethylase activity (r=0.92) and with the increase in liver weight (r=0.78) and DNA synthesis (r=0.78). Although there is a strong correlation between increase in liver weight and DNA content (r=0.999), neither of these correlated with ethylmorphine N-demethylase activity. These findings are discussed in the light of CYP3A induction, substrate specificity and inhibition; a proposed model of human CYP3A4 based on sequence homology with CYP102, a bacterial P450 of known crystal structure, demonstrates the possible mode of interaction between substrates and inhibitors within the putative active site.

Aryl Hydrocarbon Hydroxylases↗
PubMed2000

Stability of cytochrome P450 proteins in cultured precision-cut rat liver slices.

The objective of this study was to evaluate the stability of individual, xenobiotic-metabolising, cytochrome P450 proteins in precision-cut rat liver slices cultured for up to 72 h using the multiwell plate system. This was achieved using established diagnostic probes (O-dealkylation of methoxy-, ethoxy- and pentoxy-resorufin, testosterone 2alpha-hydroxylase, debrisoquine 4-hydroxylase, aniline p-hydroxylase and lauric acid hydroxylase) and immunologically using Western blotting. All cytochrome P450 activities declined in culture, the most rapid loss occurring at about 8-12 h of culture; in all cases no detectable activity was present in the 72-h cultured slices. Isoform-specific differences in the stability of various cytochrome P450 proteins were observed, with CYP2E1 being the most stable. When cytochrome P450 expression was determined immunologically, a different picture emerged. High levels of apoprotein were retained in the slices even when activity was very low. In the case of CYP2B, apoprotein levels even increased following the culture of hepatic slices. It is concluded, that for tissue slices to become an acceptable in vitro alternative system for long-term incubations, the culturing conditions must be improved to ensure that cytochrome P450 activities are better maintained.

Animals↗
PubMed2000

Bioactivation of the carcinogen 11-methoxy-16, 17-dihydro-15H-cyclopenta[a]phenanthrene.

The title compound is a more potent carcinogen than would be anticipated from its simple phenanthrene structure lacking further D-ring conjugation. In vitro it undergoes microsomal metabolism to yield as major metabolites its 15- and 17-alcohols and its 16, 17-diol; other minor metabolites are also derived from attack at the 5-membered ring, but no evidence of aromatic oxidation is apparent. The title compound is a weak mutagen in the Ames' test with Salmonella typhimurium TA100, but only with microsomal bio-activation. The 17-ol and 16,17-diol are inactive, with or without biological activation. By contrast the 15-alcohol, a rather reactive compound, is a strong mutagen both in the presence and absence of the bio-activation system. This, therefore, may be the proximate carcinogen, and its structural analogy to the naturally occurring hepato-carcinogen safrole is noted.

Androstenes↗
PubMed2000

Fate of the mushroom hydrazine agaritine in the rat and mouse.

The fate of the mushroom hydrazine [14C]agaritine was investigated in the mouse and rat strains previously employed in carcinogenicity studies with the edible mushroom Agaricus bisporus. Agaritine was rapidly absorbed in both species, achieving higher blood levels in the mouse, but with similar area under the curve. Covalent binding of agaritine material to proteins was detected only in the liver and kidney, but the extent of binding was the same in the rat and mouse. Most of the radioactivity was excreted during the first 24 hours in both animal species: in the rat it was distributed equally between urine and feces, whereas in the mouse more of the radioactivity was excreted in the urine. No qualitative differences in the metabolic profile were evident, but quantitative differences were observed. Treatment of the urine with deconjugating enzymes did not reveal the presence of any conjugates. Agaritine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzene diazonium ion were not detected in the urine or in the plasma of either species. No mutagens or promutagens were detected by the Ames mutagenicity assay in the urine of either species after exposure to agaritine. Repeated administration of agaritine to rats and mice did not alter the urinary metabolic profile and excretion of radioactivity. Similarly, feeding mice a raw mushroom diet, according to the protocol employed in the carcinogenicity studies, did not modulate the excretion of radioactivity or the urinary metabolic pattern. No major species differences in the fate of agaritine in rat and mouse were noted that could provide a rationale for the carcinogenicity of A. bisporus in the mouse, but not in the rat.

Agaricus↗
PubMed2000

Differential modulation of the genotoxicity of food carcinogens by naturally occurring monomeric and dimeric polyphenolics.

Naturally occurring dimeric polyphenolics and their gallate esters were isolated from grape seeds, almond fruits, and apple skin, and their ability to modulate the mutagenicity of food carcinogens was studied in the Ames test, and compared to that of the monomeric green tea flavonols, (+)-catechin and (-)-epicatechin. Neither the monomeric nor the dimeric polyphenols and their galloylated derivatives influenced the mutagenic activity elicited by the indirectly acting food carcinogens benzo[a]pyrene and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), in the presence of a hepatic activation system derived from Aroclor 1254-treated rats; the only exception was the B7 dimer, which, at concentrations above 1 microM, suppressed the mutagenicity of IQ. None of the polyphenolics modulated the mutagenic activity elicited by the directly acting carcinogen N'-methyl-N'-nitro-nitrosoguanidine (MNNG). In contrast, all the dimeric polyphenols and the galloylated metabolites, at concentrations over 1 microM, potentiated the mutagenic activity induced by the indirectly acting carcinogen N-nitrosopyrrolidine, in the presence of an activation system derived from isoniazid-treated rats. In conclusion, dimeric polyphenols and galloylated derivatives of plant origin are unlikely to influence the initiation stage of the carcinogenicity of chemicals through mechanisms that involve inhibition of their cytochrome P450-mediated bioactivation or scavenging of the reactive, genotoxic intermediates.

Animals↗
PubMed1999

Proliferation of hepatic peroxisomes in rats following the intake of green or black tea.

Rats maintained on green, black or decaffeinated black tea (2.5%, w/v) as their sole drinking fluid displayed higher hepatic CN- insensitive palmitoyl CoA oxidase activity than controls; the extent of increase was similar with the three types of tea. Morphological examination of the liver using electron microscopy revealed an increase in the number of peroxisomes in the tea-treated animals. The same treatment of the animals with green and black tea resulted in a similar rise in hepatic microsomal lauric acid hydroxylation. Analysis by HPLC of the aqueous tea extracts employed in the current study showed that the total flavanol content of the green variety was much higher than the black varieties, and confirmed the absence of caffeine in the decaffeinated black tea. It may be concluded from the present studies that neither caffeine nor flavanoids are likely to be responsible for the proliferation of peroxisomes observed in rats treated with tea.

Animals↗
PubMed1999

Evidence for the presence of a microsomal NADH-dependent enzyme system that can bioactivate aromatic amines in the liver of rats and mice.

Experimental evidence is presented for the presence in the liver of rats and mice of an Aroclor 1254-inducible, NADH-dependent enzyme system that can catalyse the bioactivation of aromatic and heterocyclic amines to genotoxic metabolites. It differs from the established microsomal cytochrome P450 and flavin monooxygenase systems in its response to treatment with cytochrome P450 inducing agents, optimum protein concentration and in vitro modulation by DMSO. The mutagenic metabolites generated by the NADH-supported system appear to be similar to those generated by the NADPH-mediated systems. Mutagenicity of the aminocompounds in the presence of either cosubstrate was less pronounced in an O-acetyltransferase-deficient bacterial strain, implying the presence of hydroxylamines. Moreover, glutathione potentiated the mutagenic response of both the NADH- and NADPH-generated metabolites. Cytochrome c suppressed markedly the NADPH-dependent mutagenicity of aromatic amines but had no such effect in the presence of NADH. Similarly, antibodies to cytochrome P450 reductase markedly inhibited the NADPH-, but not the NADH-dependent bioactivation of the aromatic amine 2-aminoanthracene. The cytochrome P450 suicide inhibitor, 1-aminobenzotriazole, decreased the mutagenicity of both, the NADH- and NADPH-mediated bioactivation of the aminocompounds. The above findings raise the possibility that a cytochrome P450-like protein, that can receive electrons from NADH, possibly through cytochrome b5 reductase, is present in the hepatic microsomes of rats and mice, and is capable of catalysing the bioactivation of aromatic amines through N-hydroxylation. Such a hypothesis is supported by the findings that NADH could support the O-dealkylation of 7-methoxy- and 7-ethoxy-resorufin, in the absence of NADPH. Finally the NADH-dependent bioactivation of aromatic amines was induced markedly by Aroclor 1254 and benzo(a)pyrene in Ah responsive, but not Ah nonresponsive, mice indicating that it is associated with the Ah locus.

Animals↗
PubMed1999

Consumption of tea modulates the urinary excretion of mutagens in rats treated with IQ. Role of caffeine.

The present study was undertaken to investigate whether the consumption of green tea and black tea influences the excretion of mutagens and promutagens in rats treated orally with the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Rats were maintained on aqueous extracts (2.5%, w/v) of green tea, black tea or decaffeinated black tea as their sole drinking liquid. After 4 weeks, the animals received, by gastric intubation, a single dose of IQ (5 mg/kg), and urine was collected for 48 h. Direct and indirect mutagenicity, in the presence of an activation system derived from Aroclor 1254-treated rats, was determined in the urine samples using the Ames mutagenicity assay. Consumption of green tea and black tea, but not of decaffeinated black tea, markedly decreased the urinary excretion of mutagens and promutagens. In a further study, supplementation of decaffeinated black tea with caffeine suppressed the excretion of mutagens and promutagens in the urine of rats pretreated with IQ. It is concluded that both green tea and black tea modulate the bioactivation and metabolism of IQ, and that caffeine is largely responsible for this effect.

Animals↗
PubMed1999

Reconstruction for large defects of the scalp and cranium.

Thirty-one patients with large scalp and cranium defects were reconstructed with free tissue transfer and scalp flaps. Twenty-eight of the defects resulted after tumour resection, two after infection and one after an extensive burn. Thirty-one free flaps (mainly the latissimus dorsi myocutaneous and the radial forearm fasciocutaneous) and five large (> 200 cm2) scalp flaps were used. Major complications occurred in one patient who developed meningitis and finally died (3.2%), and in one patient who lost a latissimus dorsi flap (3.2%). There were no other significant problems with the remaining patients. All other free flaps and scalp flaps survived. Primary reconstruction with free tissue transfer was our first choice of treatment because of the wide spectrum of advantages of free flaps. However, in a limited number of cases there was an indication for large scalp flaps, which, when properly designed, gave satisfactory results.

Adult↗
PubMed1999

Structural and functional integrity of precision-cut liver slices in xenobiotic metabolism: a comparison of the dynamic organ and multiwell plate culture procedures.

1. Objectives were two-fold: (1) to compare the viability of precision-cut liver slices in two culture systems, namely the dynamic organ and the multiwell plate; and (2) to evaluate whether increasing the number of slices per incubation results in a proportional increase in the extent of metabolism. 2. With both culturing systems, the major products of 7-ethoxycoumarin metabolism were the sulphate and glucuronide conjugates of 7-hydroxycoumarin with very low levels of the free compound. When the multiwell plate procedure was used, metabolism increased linearly for at least 10 h, whereas it tended to plateau after 6 h in the dynamic organ culture system. At preincubations > 10 h, significantly more metabolism of 7-ethoxycoumarin was seen in the slices cultured using the multiwell system compared with the dynamic organ system. 3. Morphological evaluation employing light and electron microscopy revealed that liver slices incubated using the multiwell system were structurally better preserved compared with those incubated using the dynamic organ system. 4. Using the multiwell system, increasing the number of slices per incubation from one to two resulted in only a modest increase in the metabolism of 7-ethoxycoumarin. The rate of metabolism of this substrate was much higher with one liver slice when expressed per mg homogenate protein. 5. It is concluded that (1) the multiwell plate culture system for culturing slices is superior to the dynamic organ system in studying the metabolism of xenobiotics following long-term incubations, (2) increasing the number of slices per incubation does not result in a corresponding increase in the rate of metabolism, and (3) in both culture systems optimal viability appears to be within 24 h of incubation.

Animals↗