Aminadav Yawetz

The effects of aroclor 1254 and petrochemical pollutants on cytochrome P450 from the digestive gland microsomes of four species of Mediterranean molluscs

Abstract 1. Clear and significant increase in cytochrome P450 content, was recorded for the Mediterranean bivalve Donax trunculus and the gastropod Avicularia gibbosula after accidental pollution of their habitat by oil spill. 2. The significant increase in cytochrome P450 content in Donax trunculus from polluted sites or after treatment with Aroclor 1254, was not accompanied by an increase, but rather a drastic decrease, in 7-ethoxyresorufin O-deethylase (EROD) catalytic activity. 3. Immunoblotting, using monoclonal antibody (1-12-3) against scup cytochrome P450E, failed to reveal the existence of a hemoprotein of the P450IA1 gene family, in Donax trunculus or Patella caerulea collected from polluted sites or treated with Aroclor 1254.

Toxicological aspects associated with the ecology of Donax trunculus (Bivalvia, Mollusca) in a polluted environment

This study provides data on the ecology and toxicology observed in the population of Donax trunculus, a sand dwelling mussel, in the shallow subtidal of Haifa Bay (Mediterranean Sea, Israel). The studied population of the mussel forms a dominant fraction in a community of sand-dwelling molluscs in a zone located 5–25 m from the shoreline, and at depths of 20–120 cm, numbering up to 2000 per m2. Samples of the mussel were collected from three sites, located within 9 km of shore in Haifa Bay. These included a clean site (Akko), a site polluted by a chemical PVC-polymer industry (Frutarom), and a site polluted with oil and waste from the petrochemical industry (Qiryat Yam). Metal analysis indicated site-dependent variations in levels of cadmium, lead, copper and mercury in the mussel soft tissues. Copper levels were similar in the bivalves collected from all the sites. Levels of mercury were significantly higher in specimens from the PVC-polluted site (Frutarom) while levels of cadmium were higher in Akko and Qiryat Yam compared to Frutarom. Lead residues were found only in Donax from Akko. The residual contents of mercury, copper and cadmium were relatively high in the young and noticeably low in Donax of medium body size. The main site of deposition of metals was in the soft tissues of the bivalve, but bioaccumulation of metals was also found in the shells. A marked increase in permeability of gills and mantle to the fluorescent anionic dye — fluorescein (FLU) was detected in Donax from Qiryat Yam and especially Frutarom, compared to the bivalves sampled from Akko. Multiple foci of enhanced permeability (multiple fluorescent spots) were detected in all the individuals sampled from Frutarom but none in the bivalve samples from Akko. Lysosomal accumulation of the metachromatic fluorescent cationic probe, acridine orange — (AO), was significantly decreased in the tissues of D. trunculus from polluted sites, especially polluted by the PVC factory. This decrease correlated with lysosomal enlargement and the formation of secondary lysosomes. D. trunculus appears to possess the most effective biochemical and physiological defense mechanisms enabling it to survive in habitats of polluted shallow waters, where other sand dwelling mollusc species were absent or found only in waters deeper then 2.5 m.

Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide.

Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.

Interaction between the δ-endotoxin produced by Bacillus thuringiensis ssp. entomocidus and liposomes

The δ‐endotoxin produced by Bacillus thuringiensis ssp. entomocidus induced the release of encapsulated [14C]sucrose from reverse‐phase vesicles composed of phosphatidylcholine and cholesterol. No such release was detected when the phospholipid component of the vesicles was either phosphatidylethanolamine, phosphatidylglycerol, or sphingomyelin. The toxin‐induced release was competitively inhibited by negatively charged organic ions while positively charged organic ions, apart from choline chloride, had no such effect. The existence of a polar head group in the phospholipid as well as intermolecular hydrogen bonding at the membrane surface, was found to be of major importance in the toxin‐liposome interaction.

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.

Metabolism of parathion and brain cholinesterase inhibition in four species of wild birds

Abstract Rates of activation and degradation of parathion by the microsomal fraction and the 12,000g supernatant of cell-free preparations were measured in four species of wild birds. Cholinesterase activity and K i values for cholinesterase inhibition by paraoxon were also determined. Cytochrome P -450 content was lowest in the African bulbul and house sparrow, intermediate in the barn owl, and highest in the blackbird. Microsomal fractions of the bulbul and the sparrow metabolized parathion to paraoxon and p -nitrophenol at higher rates than those of the blackbird and the barn owl, but arylesterase activity was lower in the two former species than in the latter two. Cholinesterase activity was lowest in the barn owl which also exhibited the lowest K i value toward cholinesterase inhibition by paraoxon. These manifestations coupled with the high rate of arylesterase activity might afford the barn owl and the blackbird with a potential defense mechanism against the toxic action of parathion and related compounds.

Cytochromes P-4501A, P-4503A and P-4502B in liver and heart of Mugil capito treated with CYP1A inducers

Hepatic microsomes of Aroclor 1254-treated Mugil capito showed a single protein band detected in immunoblot with monoclonal antibody 1-12-3 to teleost (scup) CYP1A. The hepatic CYP1A like protein was induced with dose dependency after exposure of the fish to β-naphthoflavone (BNF) as well as to Aroclor 1254. The induced mullet hepatic CYP1A protein was confined to a distinct fraction obtained by DE-52 anion exchange chromatography, and its relative content in that fraction increased in fish that were treated with higher doses of inducer. EROD (7-ethoxyresorufin O-deethylase) activity in hepatic microsomes from mullet treated with various doses of BNF correlated significantly (r(2)=0.81502, P<0.01) with CYP1A content. Treatment of the mullet with low dose of Aroclor 1254 (25 mg/kg) induced only traces of CYP1A in liver microsomes (5.1±4.8 mg/kg). However, in mullet treated with the high dose of Aroclor 1254 (100 mg/kg) there was a dramatic induction in CYP1A content (408±275 pmol/mg) and this hemoprotein comprised about 83% of the total P-450 content of liver microsomes. The total level of P-450, although induced in the liver tissue, was not induced in heart tissue of Aroclor 1254 treated mullet. On the other hand, P-4501A was induced in treated mullet to a level that comprised almost all of the cardiac P-450 content. EROD activity in the heart tissue of induced mullet was characterized by low V(max) and high K(m) values (K(m)=2.35 mM, V(max)=39.5 pmol/min per mg) compared to the values recorded for the enzyme from the liver (K(m)=1.0 mM, V(max)=288.0 pmol/min per mg). Cardiac CYP1A with low catalytic activity and repression of CYP-types other then CYP1A in heart of CYP1A induced fish may be part of a mechanism aimed to preserve crucial levels of electron donors and molecular oxygen in cardiac muscle of fish exposed to CYP1A inducers.

Components of the electron transport system in the microsomal mixed-function oxidase system in wild birds

Abstract Notable differences were found among six species of wild-caught birds in the levels of cytochrome P-450, cytochrome b5, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase. Ethyl isocyanide difference spectra showed significant variations among the species in peak height and in the ratios of the 430 455 - nm peaks. Substantial aldrin epoxidase activity was found in all species, and the amounts of dieldrin produced compared favorably with pigeon and rat liver microsomes. Higher content of cytochrome P-450 was not always accompanied by a similar rise in specific catalytic activity. Thus, no correlation could be established between these two parameters. Aldrin epoxidase activity with NADH as the sole electron donor was 25–49% as effective as with the NADPH-generating system. Addition of both NADH and NADPH-generating systems to the incubation mixture produced a synergistic effect with liver microsomes of two species but not with two other species. DDE and polychlorinated biphenyls residues were found in the heart tissue of all species examined, and this might indicate a possible inductive effect on the microsomal mixed-function oxidase system by environmental contaminants.

A new sensitive method for determining the toxicity of a highly purified fraction from δ-endotoxin produced by Bacillus thuringiensis var. entomocidus on isolated larval midgut of Spodoptera littoralis (Lepidoptera, Noctuidae)

Abstract The purification procedure of the toxic fraction from the δ-endotoxin produced by Bacillus thuringiensis var. entomocidus was considerably shortened and simplified by combining the solubilization in a high p H (10.0) solution with glycine, opening SS bonds with dithiothreitol, and releasing hydrophobic connections with the detergents Triton N-101 and sodium cholate. The subsequent fractionation was immediately continued (without dialysis or concentration) on a Sepharose 6B column, yielding the active fraction designated C. Fraction C was then passed through an octyl Sepharose 4B column, yielding the active fraction designated I, followed by gel filtration on a Sepharose 6B column yielding the active fraction designated CI. All treatments were done with the same buffer solution at 4°C. Column elution was with the same buffer but with 0.075% detergents. The purity of the fraction CI was apparent by its appearance as a well-defined band on polyacrylamide gel isoelectric focusing at p H 6.1, accompanied by a small faint band. A sensitive method for evaluating the toxicity of endotoxin fractions on an isolated midgut system (from larvae of Spodoptera littoralis ) was developed. The system is based on measuring the activity of the enzyme, reduced glutathione S -transferase, released to the medium from epithelial cells ruptured by the toxin. Fraction CI, M r 64,000, was toxic to the epithelial cells of the isolated midgut in the absence of the peritrophic membrane. When the detergents were removed by dialysis, the active protein formed high molecular weight aggregates due to hydrophobic interactions. The passage of those aggregates to the sensitive epithelial cells of the midgut was prevented by the peritrophic membrane.

Cytochrome P-450 mediated metabolism of progesterone by adrenal microsomes of PCB-treated and untreated barn owl (Tyto alba) and marsh turtle (Mauremys caspica) in comparison with the guinea-pig.

1. Significant differences between species were observed in the profile of steroids produced from progesterone by adrenal microsomes as well as in the effects elicited by Aroclor 1254 on cytochrome P-450-mediated activities. 2. In the guinea-pig, the major metabolites were products of the corticosteroid pathway but products of the androgenic pathway were also detected; in the barn owl products of both pathways were also formed while in the marsh turtle only products of the corticosteroid pathway were detected. 3. The effect of Aroclor 1254 on P-450C21 activity in the turtle and barn owl was inductive in contrast to the inhibitory effect observed in the guinea-pig.