The effect of DDE and dieldrin and their parental substances (DDT and aldrin) on PGE2 and PGF2α release from bovine endometrial explants collected during 120 to 180 days of the gestational period.

Dieldrin and DDE are environmental metabolites of the organochlorine pesticides aldrin and DDT, respectively. During pregnancy, these chemicals can quickly infiltrate through the placental barrier, accumulate in amniotic fluid and fetus, and act as endocrine disruptors (EDs). The aim of this study was to investigate the effect of DDE and dieldrin and their parental substances at concentrations of 1 and 10 ng/ml on secretion of PGE2 and PGF2α from bovine endometrial explants (120-150 and 151-180 days of pregnancy) after 24 hr of incubation with EDs. The mRNA expression of COX2, PGES and PGFS and the concentrations of PGE2 and PGF2α were measured. EDs did not affect (p>0.05) COX2 gene expression, but DDT and DDE decreased (p⟨0.05) PGES expression and PGE2 secretion in the explants from 120-150 days of pregnancy. Depending on the dose, DDT and DDE increased (p⟨0.05) PGFS expression and PGF2α secretion from the explants from 120-150 days and decreased PGF2α secretion (p⟨0.05) from the explants from 151-180 days of pregnancy. Aldrin and dieldrin decreased (p⟨0.05) PGFS expression and PGF2α secretion from all explants. In summary, EDs disrupt the secretion of PGE2 and PGF2α by influencing the gene expression of PGES and PGFS.

Enantioselective behavior of alpha-HCH in mouse and quail tissues.

alpha-HCH (hexachlorocyclohexane) is chiral and can still be detected in almost all environmental media. In this study, the enantioselective behavior of alpha-HCH in mice (CD1) and quail (Coturnix japonica) was investigated and compared after a single dose of exposure. The primary nerve cell culture was conducted to evaluate the enantioselective metabolic capacity of nerve cells of mouse and quail for alpha-HCH. In various tissues of the mice and quail, the alpha-HCH concentrations showed a typical pattern of first-order dynamics after exposure. The enantiomeric fractions (EFs) in nonbrain tissues of mice decreased substantially, indicating continuous depletion of (+)-alpha-HCH in mice. Tissue-specific EF trends in quail and enantioselective degradation of (-)-alpha-HCH in quail liver were observed. These observations indicated that the dynamic changes of EFs in mice and quail were independent of concentration changes in the same tissues. In brain tissues, the enantioenrichment of (+)-enantiomer was totally independent of their concentrations in blood. The in vitro metabolism of alpha-HCH in the primary nerve cells were negligible, and the slight EF changes in primary nerve cells demonstrated that metabolism, uptake, and excretion in the brain cells would not lead to the observed dramatic enantioenrichment of (+)-alpha-HCH in the brain tissues of the two animals. The enantioselective transport across the blood-brain barrier was the primary cause for the enantioenrichment of (+)-alpha-HCH in the brain tissues.

Expression, purification and direct eletrochemistry of cytochrome P450 6A1 from the house fly, Musca domestica.

A plasmid (pCW) was modified to code for the complete sequence of house fly (Musca domestica) cytochrome P450 6A1 (CYP6A1) with only the second amino acid changed in the N-terminal portion and this plasmid was used to express the enzyme CYP6A1 in Escherichia coli cells. With the addition of delta-aminolevulinic acid and FeCl(3) to the culture, the enzyme was produced at a level about 0.25 micromol L(-1) (15mgL(-1)) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was characterized and the content of CYP6A1 in each fraction was determined by the spectroscopic method. A nearly homogenous CYP6A1 was obtained by purification with a combination of DEAE Sepharose fast flow and hydroxyapatite chromatography. Direct electrochemistry of CYP6A1 in a didodecyldimethylammonium bromide (DSAB) film on an edge-plane pyrolytic graphite electrode (EPG) has been obtained and the catalytic activity of the enzyme to aldrin has been demonstrated by the cyclic voltammetry.

Brain regional gamma-aminobutyric acid (GABA) and locomotor activity: effect of long-term aldrin exposure.

Aldrin-induced stimulation of locomotor activity (LA) under nontolerant conditions was restored to control value after 20 or more consecutive days of aldrin administration. In contrast to the inhibition of GABAergic activity with aldrin under short-term conditions as observed in our previous study, the measurement of steady-state level of GABA, the activities of its metabolizing enzymes, turnover and the specific binding of GABA to its receptor in different regions of the brain of rats treated with aldrin (2 or 5 mg/kg/day, po) under long-term (for 30 consecutive days) conditions showed no change in the GABAergic activity in any regions of the rat brain. Moreover, the studies of the interaction between neurotransmitters (using agonist(s) and antagonist(s) of the respective neurotransmitter receptors) showed that long-term administration of aldrin restored the LA to control value by upregulation of central GABAergic activity through the interaction with dopaminergic and cholinergic systems. Thus, this result suggests that long-term aldrin exposure upregulated the central GABAergic activity inhibition under short-term aldrin treatment which may be a cause of restoration of LA stimulated by the short-term aldrin administration to its control value.

Cyclodiene organochlorine insecticide-induced alterations in the sulfur-redox cycle in CHO-K1 cells.

The effect of the cyclodiene organochlorine pesticides aldrin, dieldrin and endosulfan was assessed on CHO-K1 cultures at fractions of their lethal doses, determined by the neutral red (NRI) incorporation assay (NRI6.25, NRI12.5 and NRI25). Glutathione peroxidase, reductase and S-transferase, and total and oxidised glutathione were evaluated along the standard growth curve of the cultures. After a 24-h incubation with each insecticide, glutathione peroxidase incurred a large increase, while glutathione reductase and S-transferase activities were slightly higher than untreated controls. Unlike oxidised glutathione, the content of total glutathione declined significantly after exposure to cyclodiene insecticides. Changes in cell membrane integrity were assessed by the lactate dehydrogenase (LDH) release assay and lipid peroxidation for a wide range of pesticide concentrations. Membrane leakage and peroxide production were significantly enhanced at concentrations of aldrin and as low as 12.5 microg/ml, whereas dieldrin and endosulfan increased membrane fragility at much higher concentrations.

Aldrin-induced stimulation of locomotor activity and brain regional glutamate.

Single administration of aldrin (2-10 mg/kg) to adult male albino rats (120-130 g) enhanced locomotor activity (LA), with the maximum effect reached 2 h after treatment. The measurement of steady state levels of glutamate, glutamine and the activities of their metabolizing enzymes in different regions of the brains of rats treated with aldrin under its nontolerant condition showed that aldrin enhanced the activity of the neuronal glutamate system in the cerebral cortex, cerebellum and hypothalamus. Moreover, treatment with the glutamatergic NMDA receptor antagonist D,L-2-amino-7-phosphonoheptanoic acid, in the absence and presence of aldrin, reduced the LA of control rats and attenuated the aldrin-induced increase in LA of treated rats. These results suggest that aldrin-induced activation of the central glutamate system may be a cause of stimulation of LA with aldrin under its nontolerant condition.

Role of brain regional GABA: aldrin-induced stimulation of locomotor activity in rat.

Aldrin, a chlorinated hydrocarbon group of pesticide, is a well known central nervous system (CNS) stimulant. The CNS stimulating effect of aldrin is manifested in the form of an increase in locomotor activity (LA) of animals. Maximum increase in LA was observed at 2 h following aldrin (2-10 mg/kg, p.o.) treatment and this aldrin-induced increase in LA attained a peak at a dose of 10 mg/kg, p.o. Administration of aldrin (2 or 5 mg/kg/day, p.o.) enhanced LA of rats and reached a maxima after 12 consecutive days of treatment following which aldrin-induced LA was gradually reduced and restored to control value after 20 consecutive days of aldrin treatment. A single administration of aldrin (2-10 mg/kg, p.o.) reduced the GABA system in cerebellum, hypothalamus and pons-medulla. The treatment with aldrin (2 mg/kg/day, p.o.) for 12 consecutive days produced more inhibition in those brain regional GABA system than that observed with a single dose of aldrin. These results, thus, suggest that aldrin-induced inhibition of central GABA may be a cause of stimulation of LA with aldrin either at a single dose or for 12 consecutive days.

Aldrin-induced locomotor activity: possible involvement of the central GABAergic-cholinergic-dopaminergic interaction.

Aldrin (5 mg/kg/day, p.o.) under nontolerant condition, administered either for a single day or for 12 consecutive days, enhanced locomotor activity (LA) of rats. The increase in LA was greater in rats treated with aldrin for 12 consecutive days than that observed with a single dose. The aim of the present study is to evaluate the involvement of possible interactions of central GABAergic, cholinergic and dopaminergic systems using their agonist(s) and antagonist(s) in the regulation of LA in aldrin nontolerant rats. Administration of either L-DOPA along with carbidopa or bicuculline potentiated aldrin-induced increase in LA under nontolerant condition as well as LA of the control rats. Treatment with muscimol, haloperidol, atropine or physostigmine all decreased the LA of both aldrin nontolerant and control rats. Further, the application of (a) haloperidol along with bicuculline, atropine or physostigmine and (b) physostigmine along with bicuculline or L-DOPA + carbidopa significantly reduced LA but L-DOPA + carbidopa along with atropine or bicuculline increased LA of the control rats. These agonist(s)/antagonist(s)-induced decrease or increase in LA of the control rats were attenuated or potentiated, respectively, when those agonist(s)/antagonist(s) under abovementioned condition were administered to aldrin nontolerant rats. The attenuating or potentiating effects of aldrin on agonist(s)/antagonist(s) (either individually or in different combinations)-induced change in LA were greater in rats treated with aldrin for 12 consecutive days than that observed with a single-dose aldrin treatment. These results suggest that aldrin, under nontolerant condition, reduces central GABAergic activity and increases LA by activating dopaminergic system via inhibition of cholinergic activity. The treatment with aldrin for 12 consecutive days produces greater effect than that caused by a single-day treatment.

Indications for the involvement of a CYP3A-like iso-enzyme in the metabolism of chlorobornane (Toxaphene) congeners in seals from inhibition studies with liver microsomes.

The different isoforms of the cytochrome P450 (CYP) system can metabolise a suite of classes of lipophilic, anthropogenic compounds. The bioaccumulative potential as well as the toxicity of xenobiotics may be significantly altered in the process. To compare the metabolic ability of different wildlife species, it is important to identify the different iso-enzymes of CYP, which are responsible for the metabolism of different classes of compounds. This can be achieved with in vitro incubation assays. In the present study, preparations of hepatic microsomes of a harbour seal (Phoca vitulina) and a grey seal (Halichoerus grypus) demonstrated that the chlorobornane (CHB) congeners CHB-32 and -62 were metabolised enzymatically to their hydroxylated derivatives. These derivatives were partially characterised by their NCI mass-spectra. Inhibition studies were carried out to identify the specific CYP isoform(s) responsible for the metabolism of CHB-32 and -62. Ketoconazole has been shown to inhibit CYP3A enzymes in human and rat studies. In this study, ketoconazole caused concentration-dependent inhibition of metabolism of CHB-32 and -62, reaching 80% at the 1.0 microM treatment level. Ellipticine (1.0 microM), which has been shown to inhibit CYP1A1/2, also inhibited CHB-32 and -62 metabolism in the microsomes of grey seal, but to a much lower degree of less than 10 and 24%, respectively. In the same experiment the metabolism of 4,4'-dichlorobiphenyl was already inhibited 70% by ellipticine treatment at the same concentration. This non-ortho substituted PCB congener can easily attain a planar molecular configuration, and therefore served as a model CYP1A substrate. Inhibition of chlorobornane metabolism was not observed after the addition of goat anti-rat CYP2B antibodies or Aldrin, which is a model CYP2B substrate in rat. Cautious interpretation is advised for results obtained with so-called selective competitive inhibitors. Regardless, these studies indicated for the first time the possible involvement a CYP3A isoform in the mediation of chlorobornane metabolism in seals. The immunochemical cross-reactivity of mouse, rabbit or sheep anti-rat antibodies in the hepatic microsomes of harbour seal confirmed the presence of CYP1A1/2, CYP1A1, CYP2B1/2, CYP3A and CYP4A isoenzymes. Enantioselective metabolism by the microsomes of harbour seal was observed for both CHB-32 and -62. Stereochemical preferences of biotransformation enzymes can have an influence on the environmental distribution of both enantiomers of optically active compounds.

Six high-priority organochlorine pesticides, either singly or in combination, are nonestrogenic in transfected HeLa cells.

There have been increasing concerns that environmental chemicals may adversely affect the health of humans and wildlife by acting as endocrine modulators. These concerns have been augmented by the realization that human exposure occurs not just to single chemical agents, but typically to mixtures of chemicals that could exhibit estrogenic activity qualitatively and/or quantitatively different from that of individual components. To address these concerns, we have evaluated the ability of six organochlorine pesticides (4, 4'-DDT, 4,4'-DDD, 4,4'-DDE, aldrin, dieldrin, or endrin, all classified high priority by ATSDR) to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays, HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were exposed to these pesticides individually and in defined combinations. While estradiol consistently elicited 10- to 23-fold dose-dependent inductions in these assays, the six organochlorine pesticides showed no detectable dose-related response when tested individually. When tested in binary combinations, the pesticide mixtures showed no additional estrogenicity. Thus, the pesticides tested, singly or as mixtures, showed virtually no evidence of estrogenicity.

Induction of hepatic cytochrome P-450 by phenobarbital in semi-aquatic frog (Rana pipiens).

Hepatic microsomal cytochrome P450 (equivalent to rat P4502B1 isozymic form, a CYPIIB gene product) can be induced by pentobarbital (PB) in the adults of the semiaquatic frog, Rana pipiens (as in other terrestrial vertebrates), but not in adults of the aquatic frog Xenopus laevis or in bluegill sunfish (Lepomis macrochirus). The activity of PB-induced P450 (2B1) towards aldrin and pentoxyresorufin increases respectively by about 2- and 10-fold. This enzyme is not inducible during larval and postlarval stages of R. pipiens. However, cytochrome P4501A1 (CYPIA1 gene product) is inducible by beta-naphthoflavone in all these species. Both CYPIA and CYPIIB genes are expressed, as determined by the catalysis of their protein products, during larval, postlarval, and adult stages of R. pipiens. The concentration of P450 increases slightly during the postlarval stages until the adult stage, ready to migrate to land, is reached. This increase seems to be mostly due to 2B1-type cytochrome P450 as judged by a large increase in aldrin epoxidase but not of 7-ethoxyresorufin O-deethylase activity. It is hypothesized that the evolution of true terrestrialness, and not the evolution of air-breathing lungs alone, is required for the transcriptional activation of CYPIIB gene by PB.

The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital.

The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.

Cyclodiene insecticides-induced changes in the central depressive effect of chlorpromazine in rats.

Spontaneous motor activity (SMA), conditioned avoidance response (CAR), muscle coordination (MC) and pentobarbital sleep were tested in rats treated orally for 90 days with tolerated doses of the cyclodiene insecticides, aldrin (1 mg/kg) and endosulfan (2 mg/kg). The same tests were repeated in similarly treated animals after injecting chlorpromazine (4 mg/kg, i.p.). Both the insecticides shortened pentobarbital sleeping time indicating their microsomal enzyme inducing property. Aldrin suppressed SMA, CAR and MC, whereas endosulfan stimulated SMA, inhibited CAR and unaltered MC. However, their concurrent action with CPZ did not result in change in the central depressive effects of the latter, but its potency during the course of its action was altered. Its potency 15 min after injection was greater and 60-180 min later was lesser in these animals than that observed in control animals. This finding was interpreted to suggest that aldrin and endosulfan has quickened the biotransformation of CPZ and thereby shortened its duration of action. A temporary promotion of its potency was accounted to its active metabolites, since prior to inactivation, CPZ is known to be metabolized by the microsomal enzymes to active compounds.

Interactive effects of aldrin, cyclohexylamine, 2,4-diaminotoluene and two phorbol esters on metabolic cooperation between V79 cells.

The in vitro V79/metabolic cooperation assay measures the extent of gap-junctional transfer of metabolites from wild-type to mutant V79 cells. The assay is currently being explored as a short-term test to screen for tumor promoting chemicals, many of which inhibit metabolic cooperation. In this study, the assay was used to determine whether chemical interactions affect detection of tumor promoters in mixtures and to investigate types of interactions that may occur between chemicals. Several two-chemical mixtures were examined. The effects of phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate, two inhibitors of metabolic cooperation that operate through the same receptor-mediated pathway, were additive at concentrations below the maximally effective concentrations of either. A summation effect was observed in mixtures of two other inhibitors of metabolic cooperation, the pesticide aldrin and the principal metabolite of sodium cyclamate, cyclohexylamine. Synergistic effects were noted when PMA was combined with either aldrin or cyclohexylamine, demonstrating that chemicals in a mixture may yield a much stronger response than expected based on individual chemical exposures. Interactions were also examined between PMA, aldrin, cyclohexylamine and 2,4-diaminotoluene, a chemical that appears to enhance metabolic cooperation. 2,4-Diaminotoluene reversed effects of all inhibiting chemicals to some extent, although the pattern of response was different for each combination. In the most dramatic case, the powerful tumor promoter PMA was completely masked by 2,4-diaminotoluene. These results suggest that the V79/metabolic cooperation assay must be applied with caution in mixture testing because detection of tumor promoting chemicals can depend on other chemicals present.

Methylparathion, carbaryl and aldrin impact on nitrogen metabolism of prawn, Penaeus indicus.

Changes in hepatopancreas, muscle and gill tissue nitrogen metabolic profiles were studied in a penaeid prawn, Penaeus indicus, following its exposure to sublethal concentrations of methylparathion, carbaryl and aldrin. In all the insecticide exposed prawn tissues, Ammonia levels were significantly increased and a shift in the nitrogen metabolism towards the synthesis of urea and glutamine was observed. Inhibition of glutamate oxidation to ammonia and alpha-ketoglutarate by glutamate dehydrogenase suggests a mechanism whereby hyperammonemia is reduced by minimizing the addition of further ammonia to the already existing elevated ammonia pool. Increased alanine and aspartate aminotransferases demonstrates the onset of gluconeogenesis. Mechanisms to detoxify the ammonia by enhancing the synthesis of urea and glutamine at the cellular level was observed in the selected tissues pave way for the survivability of prawns in insecticide polluted environs.

In vivo and in vitro effects of aldrin on rat brain synaptosomal Mg2+ and Na+,K(+)-adenosine triphosphatase.

Aldrin, a chlorinated hydrocarbon, inhibited rat brain synaptosomal membrane-bound Na+,K(+)-adenosine triphosphatase (ATPase) and Mg2(+)-ATPase activities under in vivo and in vitro conditions. Na+,K(+)-ATPase was non-competitively inhibited whereas Mg2(+)-ATPase was inhibited uncompetitively. Arrhenius plots of both these ATPases without aldrin under in vivo and in vitro conditions were found to be linear. In the presence of aldrin, on the other hand, Arrhenius plots of the same ATPases were nonlinear. Slopes of Arrhenius plots of both ATPases under in vivo and in vitro condition were changed with change in temperature with aldrin. The activation energy (AE) of Na+,K(+)-ATPase and Mg2(+)-ATPase activities were changed over the temperature range 15-40 degrees in the presence of aldrin. These results thus suggest that aldrin increases the lipid fluidity of the synaptosomal membrane which may be a cause of inhibition of neuronal membrane-bound Na+,K+ and Mg2(+)-ATPase activities.

Effects of three pesticides on MSX-induced ammonia photoproduction by the cyanobacterium Nostoc linckia.

Three pesticides (2,4-D, basalin, aldrin) inhibited L-methionine-DL-sulfoximine (MSX)-induced photoproduction of ammonia by the nitrogen-fixing cyanobacterium Nostoc linckia. Combinations of pesticides and MSX were inhibitory except at low concentrations (100 and 500 micrograms/ml) of 2,4-D which stimulated NH+4 production. Similar results were obtained when pesticides were added 6 hr after the addition of MSX, but the inhibition was weaker. When MSX was added to the culture 6 hr after the addition of pesticides, the pesticides stimulated NH+4 photoproduction. Similar results were obtained on nitrogenase activity of the organism.

Effect of aldrin on accessory sex glands and plasma testosterone levels in rats.

The effects of aldrin, an organochlorine insecticide, on accessory sex glands and plasma testosterone levels in rats were studied. The aldrin was administered i.p. for 13 days and 26 days at a dose of 150 micrograms/kg. Relative weights of prostate, seminal vesicles and coagulating glands were significantly decreased in the treated rats compared to those in controls. In addition, there was a significant fall in acid phosphatase activity in prostate and fructose content in accessory sex glands was also observed in treated animals. Plasma testosterone values showed a decrease with the duration of treatment. HCG supplementation with aldrin treatment prevented all those untoward effects of aldrin in experimental rats.