J.T. Borlakoglu

Interaction of hydrophobic organic compounds with mercury adsorbed dioleoylphosphatidylcholine monolayers.

The interaction of hydrophobic organic compounds with biological membranes is an important factor in their biological activity. A fundamental insight into the mechanisms can be obtained by examining the influence of these substances on model membrane systems. The effect of four groups of hydrophobic aromatic compounds on a mercury-adsorbed dioleoylphosphatidylcholine (DOPC) monolayer is described in this paper. The compounds studied were: (1) polynuclear aromatic hydrocarbons (PAH), (2) polychlorinated biphenyls (PCB), (3) neurotoxic pesticides, and (4) phenothiazines. The monolayer properties were measured using phase-sensitive a.c. voltammetry and cyclic voltammetry. The response of the monolayer to these compounds is recorded as a change in the form of the capacity-potential curve especially with respect to two capacity peaks which correspond to two well-defined phase transitions. Planar aromatic molecules cause a negative shift of the capacity peaks, however, as the molecule becomes more globular the response becomes less and the peaks become suppressed. It is shown that planar aromatic molecules exert their effect by penetrating the hydrocarbon region of the monolayer and that there is a molecular size cut-off for higher membered PAH molecules. In addition, the monolayer is not so sensitive to the bulky PCB which have a more disruptive effect on the phase transitions and thus on the mechanisms of self-assembly of the monolayer. A direct correlation is shown between the biological membrane activity of the phenothiazines and their effect on the monolayer at submicromolar levels. A molecular selectivity of phospholipid monolayers to these compounds is indicated which has implications for their effect on biological membranes.

Expression of p450 isoenzymes during rat liver organogenesis

1. The expression of P450 isoenzymes in foetal and neonatal hepatic microsomes was determined by measuring the metabolism of marker substrates and by studying the expression of P450 isoenzymes at the protein and mRNA level. 2. Monooxygenase activities were not measurable at day 10 of gestation, but shortly before birth (day 20 of gestation) and thereafter a surge in monooxygenase activities was observed using ethoxyresorufin, aniline, nitroanisole, aminopyrine, dimethylnitrosamine and aldrin as substrates. 3. In contrast, as early as day 10 of gestation, post oxidative drug metabolism was measurable, when assessed for reactions catalysed by UDP-glucuronyltransferase, glutathione S-transferase and epoxide hydrolase. 4. Microsomal proteins isolated from foetal/perinatal rats did not crossreact with antibodies raised to CYP1A1, CYP1A2, CYP2A1, CYP2B1, CYP2E1, CYP3A1 and CYP4A1 at a protein loading of 3 micrograms total protein/well. 5. With the exception of CYP2E1 mRNA and CYP4A1 mRNA there was little evidence to suggest the expression of CYP1A1, CYP1A2 and CYP2A1 mRNA. 6. The mRNA of CYP2B1, CYP2C7 and CYP3A1 was not detectable in foetal/perinatal rat liver extracts at a loading rate of 10 micrograms total RNA. 7. Microsomal proteins isolated from neonatal rats crossreacted with antibodies raised to CYP2C6, CYP2E1, CYP3A1 and CYP4A1, albeit at varying intensities. 8. Concomitantly, CYP2A1, CYP2E1 and CYP4A1 mRNA transcripts were detectable in Northern blot hybridization experiments using neonatal rat liver RNA extracts.

Polychlorinated biphenyls in fish-eating sea birds—molecular features and metabolic interpretations

Abstract Polychlorinated biphenyls (PCBs) are an important group of environmental pollutants, there being a total of 209 theoretical congeners. Residue analysis of the adipose tissue of five species of fish-eating sea birds from British and Irish coastal waters revealed the presence of up to 60 different congeners. By GC-MS, GC-MSD and high resolution capillary GC-ECD using authentic standards, it was possible to identify and quantify 40 different congeners. Despite the large number of PCB congeners identified only 10 accounted for > 80% of the total PCBs. A PCB congener was identified accounting for 5% of the total PCBs (2,3′,4,4′,5′,6-hexachlorobiphenyl) [168] not usually reported in biological samples. Comparison of the molecular structure for the persistent PCB congeners revealed the lack of meta-para unsubstituted adjacent carbon atoms. It has been shown that meta-para unsubstituted adjacent carbon atoms facilitate the metabolism of PCBs and it is hypothesised that the formation of hydroxy derivatives may depend upon such a requirement.

Alterations in rat hepatic drug metabolism during pregnancy and lactation

The hepatic microsomal drug metabolism during pregnancy and lactation was studied. Four days post partum, the concentrations of cytochrome P450 and cytochrome b5 were reduced by 50% when compared with pregnant rats, at day 10 of gestation. Within this time period the N-demethylation of aminopyrine, the rate of aldrin epoxidation and the N-demethylation of demethylnitrosamine was reduced by 53, 74 and 21%, respectively. However, the rates of ethoxyresorufin-O-deethylation did not differ amongst both groups and the deethylation of 4-nitroanisole and the 4-hydroxylation of aniline was increased by 71 and 31%, respectively in lactating rats. Furthermore, the activities of UDP-glucuronyltransferase and glutathione S-transferase were increased by 21 and 27%, but those of epoxide hydrolase were reduced by 85%. Western immunoblot analysis of microsomal proteins obtained from pregnant and lactating rats shows that only proteins encoded by the genes of CYP2C6 and CYP3A1 are expressed at detectable levels, whereas the expression of CYP1A1, CYP1A2, CYP2A1, CYP2B1, CYP2E1 and CYP4A1 was not detectable in pregnant and lactating rats at a protein loading of 3 micrograms total protein per well. In contrast, in northern blot hybridization experiments, detectable amounts of mRNA of the above named isoenzymes were measurable, but at varying intensities. Based on the northern blot hybridization analysis, an approximate 4-fold and 3-fold increase in CYP2A1 mRNA and CYP3A1 mRNA was found, when lactating rats were compared with female controls or pregnant rats, at day 10 of gestation.

Activity of the ethylene-forming enzyme in relation to plant cell structure and organization

Summary Activity of the ethylene-forming enzyme (EFE) has been compared in leaf discs, isolated cells, protoplasts and vacuoles derived from leaves of pea ( Pisum sativum L.) and bean ( Vicia faba L.).Some 95% of the activity observed with leaf tissue was lost when cells were isolated from the tissue.The limited activity observed with the isolated cells was largely retained by protoplasts and vacuoles isolated from the protoplasts, but was lost when the vacuoles were lysed.Light inhibited EFE activity but did not greatly affect the percentage loss of activity when cells were isolated.Isolation of cells by gentle disruption of cladophylls from Asparagus sprengeri Regel.resulted in a loss of about 80% of EFE activity.It is proposed that full EFE activity requires tissue integrity in addition to a previously noted requirement for cell membrane integrity.

Metabolism of di-, tri- and tetrabromobiphenyls by hepatic microsomes isolated from control animals and animals treated with Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs).

1. The metabolism of a wide range of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with Aroclor 1254 was studied. 2. Hepatic microsomes isolated from control rats expressed higher rates of oxidations than avians. 3. Treatment of rats and pigeons with Aroclor 1254 induced cytochrome P450 dependent monooxygenases leading to an increased regioselective metabolism of PCB isomer and congeneres. 4. There was an inverse relationship between the degree of halosubstitution and microsomal oxidation. Meta-para carbon atoms free of halosubstitution were the preferred side for oxidation. 5. A good correlation was found between the in vitro metabolism of PCBs and their relative abundance in tissue extracts, thus suggesting oxidative metabolism to be the major route of metabolic disposal.

Cytochromes P-450 of sea birds: Cross-reactivity studies with purified rat cytochromes

1. Polyclonal antibodies against rat cytochrome P-450c (IA1), P-450d (IA2), P-450b (IIB1), P-450h (IIC11) and P-450j (IIE1), were used to probe liver microsomes prepared from six sea bird species collected from the Irish Sea between 1978 and 1988. 2. Significant cross-reactivity in all the sea bird species was seen only with antibodies to P450 IA1. Expression of cross-reactivity proteins was highly variable between individual birds, which show evidence of environmental induction. 3. Shared epitopes to P450 IA1 and IA2 were seen on a single protein expressed in liver microsomes from the cormorant (Phalacrocorax carbo). 4. Antibodies against members of rat P450 gene family II showed a small degree of cross-reactivity with sea bird microsomes. Antibodies against P450 IIB1 and IIC11 showed weak cross-reactivity in all species with little inter-individual variation. Antibodies to P450 IIE1 showed no cross-reactivity in any bird species. 5. P450 gene family I appears to be well represented in sea birds while P450 gene family II is not well developed in this group of lower vertebrates.

Transplacental transfer of polychlorinated biphenyls induces simultaneously the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf

At day 15 of gestation, rats were injected with a single i.p. dose of 100, 250 and 500 mg/kg body weight of a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1254). Seven days later, significant increases in maternal and foetal cytochrome P450, cytochrome b5 and cytochrome c (P450) reductase were found. Concomitantly, the metabolism of nitroanisole, aniline, ethoxyresorufin and benzo[a]pyrene was significantly increased, but foetal metabolism of dimethylnitrosamine was not detectable and only marginal increases in the metabolism of aminopyrine and aldrin were seen. In contrast, maternal metabolism of dimethylnitrosamine, aminopyrine and aldrin was measurable, but significant increases were determined only with the latter substrate. Transplacental transfer of PCBs resulted in increased metabolism of substrates catalysed by foetal CYP1A1 and CYP2B1, but there was no evidence for CYP2E1-catalysed reactions. Further measurements show significant increases in foetal and maternal epoxide hydrolase, glutathione-S-transferase and UDP-glucuronyl transferase activities, thus suggesting that treatment with Aroclor 1254 resulted in coordinated increases in foetal and maternal oxidative and post-oxidative drug metabolism. Western blot analysis of microsomal proteins shows the induction of foetal and maternal CYP1A1, CYP1A2, CYP2A1, CYP2B1, CYP3A1 and CYP4A1. In addition, increased expression of CYP2C6 was seen with the mother but not the foetus. Unlike the mother, foetal rats did not express CYP2E1 and the expression of the above-listed P450 isoenzymes was greater in the mother than the foetus. Northern blot analysis shows significant increases in maternal and foetal CYP1A1, CYP1A2 and CYP2B1 mRNA. An increased amount of CYP3A1 mRNA was only seen with the mother, but not the foetus. Treatment of mothers with Aroclor 1254 resulted in reduced CYP2A1, CYP2C7, CYP2E1 and CYP4A1 mRNA. Insignificant differences in the expression of foetal CYP2A1 and CYP4A1 mRNA were found, but in utero exposure to PCBs reduced the amounts of CYP2E1 mRNA and there was no foetal CYP2C7 mRNA transcript. Treatment with Aroclor 1254 increased the expression of the protooncogenes c-Ha-ras and c-raf in the mother and the foetus, but at varying intensities. Pregnancy itself was linked to an increased expression of these protooncogenes. erbA and erbB mRNA was not detected.

Correlations between the molecular structures of polyhalogenated biphenyls and their metabolism by hepatic microsomal monooxygenases.

1. Collation of the data presented in the preceding papers (references 4,5) showed a significant correlation (r = 0.83; P < 0.001) between the molecular mass (and hence the extent of halosubstitution) of halogenated biphenyls and their rate of hydroxylation by hepatic microsomal monooxygenases. 2. There was no relationship between the extent of polyortho halosubstitution of biphenyl and the rate of metabolism. 3. A marginal correlation (r = 0.33; P < 0.001) was found when the number of adjacent unsubstituted meta-para positions were linked to the rate of metabolism of PCBs. This structural feature facilitates microsomal oxidation. 4. The results support the proposal that PCBs with meta-para hydrogen atoms are less enriched in tissues of animals and humans as this structural feature favours their metabolism by P450 isoenzymes.

Evidence for the induction of cytochrome P‐452 in rat liver by Aroclor 1254, a commercial mixture of polychlorinated biphenyls

We have examined the ability of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) to induce hepatic cytochrome P‐452‐linked enzyme activities in rat and pigeon liver five days after its intraperitoneal injection. The results provide evidence that, at the doses used, Aroclor 1254 induces cytochrome P‐452‐linked enzyme activities in rats, but not in pigeons. This inductive effect was previously regarded as being specific for hypolipidemic drugs and phthalate ester plasticisers.