Preparation and Characterization of Aptamers Against O,p'-DDT.

The compound 1,1,1-trichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethane (o,p'-DDT) has been identified as one of the endocrine-disrupting chemicals causing adverse effects on wildlife and even humans through bioaccumulation. Its detection has become increasingly important. We have obtained candidate aptamers binding to o,p'-DDT by a systematic evolution of ligands by exponential enrichment (SELEX) protocol. Five out of seventeen candidate sequences were selected for preliminary characterization by SYBR Green I assay. One sequence with highest fluorescence response with o,p'-DDT, designated DDT_13, was chosen for further characterization. Its dissociation constant (Kd) was determined to be 412.3 ± 124.6 nM. DDT_13 exhibited low cross-binding activities on other tested small molecules. The good bioactivities of DDT_13 were demonstrated for the analysis of spiked lake water and tap water samples. This study provides a novel o,p'-DDT-specific probe for its future applications.

In vitro effect of N-acetylcysteine on hepatocyte injury caused by dichlorodiphenyltrichloroethane and its metabolites.

The organochlorine pesticide, dichlorodiphenyltrichloroethane (DDT), is still used to combat the spread of malaria in several developing countries despite its accumulation and known hepatotoxic effects that have been demonstrated both in vitro and in vivo. N-Acetylcysteine (NAC) is a recognized hepatoprotective agent that has been reported to reduce hepatotoxicity initiated by many different compounds. The aim of this study was to determine whether NAC could counter in vitro hepatocyte injury induced by DDT or its two major metabolites, dichlorodiphenyldichloroethylene and dichlorodiphenyldichloroethane. HepG2 cell cultures were used to assess the following parameters of toxicity: cellular viability, intracellular levels of reactive oxygen species (ROS), mitochondrial membrane potential and initiation of apoptosis. None of the three test compounds induced ROS generation, yet exposure to any of the three compounds produced mitochondrial hyperpolarization, which was countered by NAC pretreatment. All three test compounds also induced apoptotic cell death, which was inhibited by NAC. Despite NAC counteracting some adverse intracellular changes due to organochlorine exposure, it appeared to aggravate the cytotoxic effects of the organochlorine compounds at low test concentrations. As the same outcome may also occur in vivo, results from the present study raise concern about the use of NAC as treatment for DDT-induced hepatotoxicity.

Immunization prevents DDT buildup in mouse tissues.

DDT is used for pest control, causing health and environmental hazards in some parts of the world. The goal of this study was to assess whether immunization against a toxic compound could reduce the toxicant uptake of an organism, specifically to develop a DDT immunization that promotes the production of specific antibodies and assess whether it reduces DDT levels in the bodies of mice that are exposed to DDT by intake. BALB/c mice were immunized with DDT-keyhole limpet hemocyanine (DDT-KLH) conjugate (n=10) or unconjugated KLH (n=10), which was used as a control. After the immunization specific DDT antibodies in the mouse serum were determined by ELISA and then the mice were fed chow containing 40 mg/kg of DDT for 45 days. Finally, the concentration of DDT and its metabolites, DDE and DDD, in various tissues was measured by gas chromatography. Specific DDT antibody levels were significantly higher in the DDT immunized group than in the control group. DDT, DDE and DDD levels in adipose tissue, blood, brain and spleen were significantly reduced in the DDT immunized animals relative to control animals. However, DDT and DDD levels were higher in the liver compared to the control group. The findings indicate that the DDT immunization reduces the total uptake of DDT in animal tissues, which is reflected by the lower levels in adipose tissue, blood, brain and spleen. The elevated levels in liver suggest that DDT-antibody complexes in mouse serum are delivered to the liver.

Resveratrol reverses tumor-promoter-induced inhibition of gap-junctional intercellular communication.

The naturally occurring stilbene/alexin trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is a promising agent for the prevention of cancer. We investigated the effect of resveratrol on gap-junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells because inhibition of GJIC is an important mechanism of tumor promotion. Seventeen to 50 microM resveratrol increased GJIC significantly by a factor of 1.3 compared with solvent vehicle controls, when the WB-F344 cells were exposed to resveratrol for 6 h. Most tumor promoters, including the phorbol ester TPA and the insecticide DDT, block GJIC. Resveratrol at 17-50 microM also significantly prevented down-regulation of GJIC by TPA and DDT, by a factor of 2.7 and 1.8, respectively. This recovery of GJIC from TPA inhibition was partly correlated with hindered hyperphosphorylation of Cx43. In conclusion, resveratrol was found to enhance GJIC and counteract the effects of tumor promoters on GJIC, and this is likely a mechanism that contributes to the antipromotional and anticarcinogenic properties of resveratrol.

The antiestrogen ICI 182,780 abolishes developmental injury for murine embryos exposed in vitro to o,p'-DDT(1).

Previously, we reported that in vitro exposure of murine embryos to 0.1 microg/ml o,p'-DDT (an estrogenic pesticide) significantly reduced development to blastocyst and mean cell number per embryo, and increased percent cell death by 96 h of culture. The objective of the present study was to determine if developmental injury induced by o,p'-DDT resulted from estrogenic, antiestrogenic, or unrelated adverse biologic mechanisms. Toward this objective, pronuclear embryos from CD-1 mice were cultured 96 h in medium supplemented with 0.1% ethanol (control) or 0.1 microg/ml o,p'-DDT, 17beta-estradiol, or ICI 182,780 dissolved in ethanol as single agents or as paired mixtures. As single agents, development to blastocyst and mean cell numbers were significantly reduced and percent apoptosis was significantly increased for embryos cultured in the presence of o,p'-DDT or ICI 182,780. Development to blastocyst was significantly reduced for embryos cultured in the presence of 17beta-estradiol. Beneficial interaction occurred when the receptor antagonist ICI 182,780 was combined with either receptor agonist (o,p'-DDT or 17beta-estradiol). In contrast, interaction was not significant when the two agonists were combined. The results indicate that developmental injury due to the estrogenic pesticide o,p'-DDT was abolished by the addition of the receptor antagonist ICI 182,780 and not by the receptor agonist 17beta-estradiol. The findings underscore the utility of the model for uncovering mechanisms of developmental injury.

Blockade of only spinal alpha 1 adrenoceptors is insufficient to attenuate DDT-induced alterations in motor function.

Male Fischer 344N rats were chronically implanted with an intrathecal cannula and gavaged with p,p'-DDT (1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane; 30 or 45 mg/kg) or corn oil. Seven hours later, subjects were intrathecally infused with vehicle, 15, 30, 60, or 120 micrograms of prazosin (an alpha 1-adrenergic antagonist). Spectral analysis of bodily movements was performed 7.5, 8, and 10 hr after DDT administration. In control rats, 15 micrograms of prazosin reduced the spectral profiles of spontaneous movements. A 30-micrograms dose produced motor impairments, without significantly changing the spectral profiles. Tremulous movements induced by DDT were unaffected by 15 or 30 micrograms, whereas 60 or 120 micrograms of intrathecal prazosin significantly reduced the spectral profiles of rats pretreated with 45 mg/kg of DDT. Other subjects were administered vehicle or DDT (45 mg/kg), intrathecally infused with 15 or 60 micrograms of prazosin (7 hr), and sacrificed (7.5 hr). Noncannulated rats were gavaged with 60 mg/kg of DDT, injected subcutaneously (sc) with 0.5 mg/kg of prazosin (5.5 hr), and sacrificed (8 hr). Cortical and spinal tissues were used in ex vivo binding assay utilizing [3H]prazosin. Fifteen or sixty micrograms of intrathecal prazosin occupied similar percentages of spinal [3H]prazosin binding sites, but produced a dose-related increase in cortical prazosin equivalents. Sixty micrograms of intrathecal or 0.5 mg/kg of sc prazosin resulted in similar concentrations of cortical prazosin equivalents. Together, these data indicate that while intrathecal prazosin will attenuate DDT-induced motor dysfunction, this effect requires blockade of alpha 1 adrenoceptors in regions other than solely the spinal cord.

[The DDT and malathion resistance of malarial mosquitoes in the Mariupol area]. / Rezistentnost' k DDT i malationu maliariinykh komarov na territorii Mariupolia.

The paper presents results of comparative studies of the level of sensitivity of endophilic malarial mosquito species to DDT and malathion. The sensitivity of the vectors was shown to remain at the same level in spite of cessation of DDT use in medical practice. It may be probably due to the use of organochlorines as pesticides within the city area. Wide use of organophosphorous preparations in medical and agricultural practices has not still led to malathion resistance.

[The sensitivity of a natural population of Anopheles martinius to malathion and DDT in the Kirghiz SSR]. / Chuvstvitel'nost' prirodnoi populiatsii Anopheles martinius k malationu i DDT v Kirgizskoi SSR.

Levels of An. martinius natural population resistance to DDT and malathion were determined in the Kirghiz SSR by a generally accepted WHO method. 100% sensitivity of mosquitoes to malathion and limited DDT-resistance (mean death rate, 59.1%) were revealed. Age-related differences in DDT-resistance level were established. Mosquitoes at the day of imago hatching were less sensitive: the death rate in case of the contact was 42.7%. Blood sucking produced no effect on their resistance.

[The level and nature of insecticide resistance (DDT and malathion) in laboratory-strain mosquitoes]. / Uroven' i priroda rezistentnosti k insektitsidam (DDT i malationu) komarov laboratornykh linii.

Authors characterize 7 mosquito strains and species from the collection of the E. I. Martsinovskii Inst. with regard to the levels and mechanisms of their resistance to malathion and DDT. Resistance mechanisms of the strains An. stephensi, An. sacharovi, An. atroparvus, Ae. aegypti and C. pipiens were determined with the help of synergists. Malathion-resistance in one of the An. stephensi strains is due to high carboxyl activity. Other strains featured various levels of DDT-resistance due to various mechanisms: in Ae. aegypti it was due to mixed-function oxidases activity; in An. atroparvus, An. sacharovi and An. stephensi second strain to glutathione-dependent transferase, in C. pipiens, An stephensi third strain and, partially, An. sacharovi,--to the probable presence of kdr-factor.

[Behavior of Anopheles mosquitoes in contact with insecticides]. / Povedenie komarov Anopheles pri kontakte s insektitsidami.

The rate of irritability and "aggressiveness" (attacking activity) at the contact with DDT was determined for all main potential vectors of malaria in the USSR. Great species differences were found out. Changes in the irritability and "aggressiveness" rates with growth of resistance to DDT were traced. The knowledge of resistance rate, initial data on the irritability and "aggressiveness" of each species and the main trends in the changes in mosquitoes behaviour under the effect of DDT makes it possible to predict the effectiveness of DDT in concrete situations. The results of visual observations on the behaviour of malaria vectors in dwelling treated with malathion and propocsur, substitutes for DDT in control on resistant populations, are given.

5,5-Diphenylhydantoin antagonizes neurochemical and behavioral effects of p,p'-DDT but not of chlordecone.

Rats were given 75 mg/kg of 5,5-diphenylhydantoin (phenytoin) or vehicle 30 min prior to 75 mg/kg of 1,1,1-trichloro-bis(p-chlorophenyl)ethane (p,p'-DDT) (p.o.) or chlordecone (i.p.) and tremor was measured 12 h later. Rats were then killed, and regional brain levels of biogenic amines and their acid metabolites and amino acids were determined. Pretreatment with phenytoin significantly attenuated the tremor produced by p,p'-DDT but enhanced that produced by chlordecone. p,p'-DDT had significant effects on the levels of aspartate, glutamate, 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), whereas chlordecone increased glycine, 5-HIAA, and MHPG levels. Pretreatment with phenytoin blocked p,p'-DDT-induced increases of aspartate in the brainstem and spinal cord, 5-HIAA in the hippocampus, and MHPG in the brainstem and hypothalamus. Phenytoin significantly enhanced chlordecone-induced increases of MHPG in the brainstem. These data indicate that organochlorine-induced increases in noradrenergic activity in the brainstem and spinal cord may be directly related to the tremorigenic effects of these chemicals.

A possible neurochemical basis of the central stimulatory effects of pp'DDT.

The striatal neurochemical changes induced by pp'DDT (600 mg/kg) in mice were: an increase in the concentration of free ammonia, a decrease in the level of GABA and a reduction in the level of acetylcholine. These changes were maximal 5 h after treatment with pp'DDT, when the animals developed 'severe' convulsions. The convulsions and striatal neurochemical changes were modified to different degrees by barbiturates. Phenobarbitone protected all the animals from pp'DDT-induced convulsions. The levels of striatal acetylcholine and GABA in these animals were within normal limits. Prominal reduced the severity of convulsions in pp'DDT-treated animals. The levels of striatal acetylcholine and GABA were significantly lower than control values in these animals. Primidone neither modified the convulsions nor the striatal neurochemical changes in pp'DDT-treated animals. The increase in the concentration of free ammonia, in pp'DDT-treated animals, was not modified by barbiturates. Aminooxyacetic acid raised the GABA level above normal and abolished the convulsions in pp'DDT-treated animals; the level of acetylcholine was within normal limits in these animals. Hydroxylamine produced a similar but less marked effect. Pyridoxine had no effect on convulsions or striatal neurochemical changes induced by pp'DDT. The increase in the concentration of free ammonia in pp'DDT-treated animals was not modified by these agents. It is likely that pp'DDT produced stimulatory effects by increasing the concentration of free ammonia which may be involved in reducing the level of GABA, while changes in the level of acetylcholine may be an effect of pp'DDT-induced convulsions.

Role of striatal acetylcholine and free ammmonia in the central stimulatory effects of pp'DDT in rats. Protective effects of barbiturates.

pp'DDT (600 mg/kg, orally) produced an increase in the level of free ammonia and a decrease of acetylcholine in the corpus striatum of rats. These effects were maximal 5 h after the administration of pp'DDT. The animals showed tremors after 2 h, mild or moderate convulsions after 3.5 h and severe convulsions 5 h after treatment with pp'DDT. The neurochemical changes and convulsions induced by pp'DDT were modified to different degrees by barbiturates. Phenobarbitone sodium abolished the convulsions while prominal reduced the severity of convulsions in pp'DDT treated animals. The level of striatal acetylcholine in pp'DDT-phenobarbitone treated animals was not significantly different from the control values. In prominal-pp'DDT treated animals, it was slightly but significantly reduced. Primidone neither changed the severity of convulsions nor the level of striatal acetylcholine in pp'DDT treated animals. Further barbiturates had no significant effect on the level of free ammonia in pp'DDT treated rats. The results suggest that changes in the level of acetylcholine are not the cause but an effect of pp'DDT induced stimulatory effects or convulsions, mediated through an increase in the level of free ammonia or some other mechanism.