Effect of in vitro administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on DNA-binding activities of nuclear transcription factors in NIH-3T3 mouse fibroblasts

Abstract

Most modern pesticides are expensive. Application of excessive dosage rates is likely to cause undesirable biological side‐effects and is economically wasteful. Non‐uniform distribution of the spray cloud, or application at the wrong time, may result in failure to control the pest. It is the responsibility of the field operator to acquire sufficient knowledge and skill to ensure proper use of the control agents, to increase efficiency of their usage and to reduce unwanted side‐effects. To achieve this goal, he must take into consideration the various physical factors that govern field performance of pesticides.

A simple relationship exists between the spray volume and emission rate used, and droplet size produced. The use of extremely low spray volumes (i.e., those less than 2.0 litre per ha) for forest insect control in Canada, as opposed to higher volumes used in agriculture, necessitates the release of fine droplets (ranging from 20 to 70 μm in diameter) to obtain adequate coverage of the target area. These droplets take a long time to sediment downwards, evaporate in‐flight, become smaller in size and/or form powdery residues, thus contributing to off‐target drift and impaired droplet adhesion to target surfaces. Physical factors such as rain washing, degradation by sunlight and erosion by wind also influence the longevity of pesticide deposits on foliage which is crucial during the critical period of pest control.

Factors affecting the mode of entry into insects are related to the type of ingredients used in formulation. If a pesticide acts via crawling contact, formulations which would provide surface deposits would be more beneficial than emulsions or oil‐based mixes which tend to undergo penetration into foliar cuticle. Physical factors that affect field performance of a pesticide tank mix are related to phase separation and ‘breakdown of emulsions’ in the application equipment; ‘agglomeration and caking’ of wettable powder dispersions at the bottom of the tank; impaired flow behaviour of highly viscous formulations; and coarse atomization of high‐viscosity tank mixes leading to poor target cover.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced down-regulation of glucose transporting activities in mouse 3T3-L1 preadipocyte

The possibility that 3T3-L1 preadipocytes, while the level of its glucose uptake activity is relatively low, may offer a useful tool for studying the cause for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced "lipolytic response" was studied. It was established first, that TCDD causes reduction of glucose uptake, one of the hallmark events of the lipolytic process. Then the function of c-Src was investigated. The antisense c-src oligonucleotide decreased the inhibitory action of TCDD on glucose uptake activity in a sequence specific manner. Since antisense oligonucleotides are known to own their blocking effects to their ability to reduce translation of proteins, Western blotting analysis was performed to verify their effectiveness. As expected, the treatment of pre-adipocytes with antisense c-src oligonucleotide reduced c-Src in a sequence specific manner. The treatment of antisense c-src oligonucleotide alone was sufficient to diminish the inhibitory action of TCDD on glucose uptake activity in 3T3-L1 cells, indicating that c-Src is somehow involved in the action of TCDD. In a similar manner, the contribution of c-Fos was investigated using antisense c-fos oligonucleotide, since c-Fos is known to be one of the most affected proteins by c-Src activation among AP-1 members. The treatment of antisense c-fos oligonucleotide did not block the effect of TCDD on glucose uptake activity in 3T3-L1 cells. Therefore, it is unlikely that c-Fos is very important in the lipolytic signal transduction of TCDD mediated through c-Src. In order to determine the relationship between c-Src and c-Myc in the mitotic signal transduction pathway, the effect of antisense c-myc oligonucleotide was investigated. Basically the same result as antisense c-src oligonucleotide experiment was obtained thereby, suggesting the importance of c-Myc as well as c-Src in the signal transduction of TCDD. To show the effect of antisense c-myc oligonucleotide treatment, the level of c-Myc protein by Western blotting and electrophoretic gel-mobility shift assay was assessed. However, antisense c-myc oligonucleotide treatment increased the activity of c-Myc in a sequence specific manner. This may be the result of cellular compensatory response to the initial suppression of c-Myc by antisense treatment. The observation that antisense c-fos oligonucleotide could not block the effect of TCDD indicates that this preadipocyte model is different from the adipocyte differentiation model.     

T cell-derived IL-5 production is a sensitive target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The immune system is one of the organs most vulnerable to the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Among the various immunotoxic effects of TCDD, the thymus involution and suppression of IgM antibody production are well known sensitive reactions of the thymocytes and B cells affected by TCDD. Recently, we reported that TCDD greatly inhibits the production of type-2 helper T (Th2) cell-derived cytokines, especially IL-5, by the splenocytes in mice immunized with ovalbumin (OVA). In the present study, we investigated the dose-dependency of these TCDD immunotoxic effects in OVA-immunized mice to identify the most sensitive target. Mice of two age groups, 6 weeks old and 3 weeks old, were dosed with 0.3, 1.0, or 3.0 microg TCDD/kg and immunized with OVA using alum as an adjuvant. Seven days later, the thymus weight, thymocyte population, antigen-specific IgM in the plasma, and IL-5 production by the splenocytes were examined. Among them, IL-5 production was significantly suppressed by all three doses of TCDD and reduced to about 30% by even a small dose of 0.3 microg TCDD/kg in both age groups. The thymus weight was significantly reduced by 1.0 microg or 3.0 microg TCDD/kg, but IgM production was not affected by up to 3.0 microg/kg of TCDD in both age groups. Taken together, the Th2 cell-derived IL-5 production was the most sensitive endpoint detecting TCDD toxicity among those examined. Our results also suggest that effector T cells are targets more vulnerable to TCDD toxicity than thymocytes or antibody-producing B cells in the OVA-immunized mice.     

Hemopoietic cell kinetics after intraperitoneal single injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely spread environmental pollutant. Homopoietic system is one of the targets of TCDD in laboratory animals including monkeys. The present study is the hemopoietic cell kinetics in mice, from the severe depression in cellularity of bone marrow and CFU-GM, to their recovery after the intraperitoneal injection of high dosage of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The bone-marrow cellularity and CFU-GM were severely decreased to 37.8% and 48% of the control, respectively until day 1 after exposure to TCDD. They were, however, soon recovered, even overshot the control value. Subsequently, they tended to show decrease and oscillation again to and under the control value. In conclusion, our cell kinetic study has proven the oscillation in bone-marrow cellularity and CFU-GM during the recovery period, of which the observation seems to be useful to extend our understanding in the hematotoxicity of TCDD.     

Inhibition of ethoxyresorufin-O-deethylase (EROD) activity in mixtures of 2,3,7,8-tetrachlorodibenzo-p-dioxin and polychlorinated biphenyls

Induction of ethoxyresorufin-O-deethylase (EROD) activity and porphyrin accumulation shows different structure-activity relationships for different polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Interactions between the two responses can strongly influence the induction and activity of EROD. The results support the conclusion that there are nonadditive interactions between nondioxin-like PCBs and dioxin-like compounds. The interaction between EROD activity and the porphyrin biosynthesis makes the prerequisite of additivity in the toxic equivalency factor concept for environmental mixtures highly spurious. Inhibition of EROD activity caused by non-dioxin like compounds could have a significant impact on the value of EROD activity as a biomarker in the present methods of risk assessment for these compounds.     

TCDD (2,3,7,8-tetrachlorodibenzo-P-dioxin) causes reduction in glucose uptake through glucose transporters on the plasma membrane of the guinea pig adipocyte.

A single dose of 2,3,7,8-TCDD (1 micrograms/kg, i.p. injection) resulted in a significant decrease in cellular 3-O-methyl-[3H]-glucose uptake by guinea pig adipose tissue and pancreas after 24 hours. An in situ tissue culture study in which pieces of adipose tissue were incubated with 10(-8)M TCDD showed a time-dependent decrease in glucose uptake. Reconstitution of adipocyte plasma membrane from tested or control animals into artificial liposomes also resulted in this difference in glucose uptake. Binding of [3H]-cytochalasin B, a specific inhibitor of glucose transporter proteins, was significantly lower in acetone-ether powder preparations of TCDD-treated adipose tissue than from controls, suggesting that the total titer of these proteins is decreased by TCDD. Finally, the relevance of these results to glucose or lipid metabolism was tested. Lipoprotein lipase (LPL) activity of guinea pig adipose tissue was decreased after 8 hours of in situ incubation with TCDD indicating that glucose uptake was depressed at an earlier time point. These findings may contribute to a better understanding of dioxin-induced "wasting syndrome".     

Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs): support for the use of the in vitro AHH induction assay

The in vitro induction of the cytochrome P₁-450-dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-TCDD and related toxic halogenated aryl hydrocarbons in rat hepatoma H-4-II E cells has been developed as a short term quantitative bioassay for these toxic chemicals. There was a linear correlation between the -log EC₅₀ (in vitro) AHH induction vs the -log ED₅₀ (in vivo) for body weight loss, thymic atrophy, hepatic AHH and EROD induction in the rat for several polychlorinated biphenyl, dibenzo-p-dioxin and dibenzofuran congeners and mixtures. These data clearly support the utility of the in vitro AHH induction assay as a short term test system for quantitating the “toxic or 2,3,7,8-TCDD equivalents” in an extract containing toxic halogenated aromatics. The bioassay method is rapid, relatively accurate and much more cost effective than conventional analytical methods such as gas-chromatography-mass spectrometry from which it is difficult to determine the levels of 2,3,7,8-TCDD equivalents in specific analytes.     

Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum

The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms’ digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms’ health and the chemical quality of aquatic ecosystems.     

Effect of environmental factors on pheromone trap catches of (Hubner)

Prediction models relating pheromone trap catches of (H) with several onvironmental factors are worked out. It is expected that those models will help overcome indiscriminate use of pesticides.     

Effect of acute exposure to PFOA on mouse liver cells in vivo and in vitro

Increasingly, epidemiological evidences indicate chemosynthetic perfluorooctanoic acid (PFOA), an environmental pollutant, induces potential adverse effect on human health after long-term exposure. However, less study has been performed for assessment of acute effect of PFOA exposure on metabolic homeostasis. In experimental designs, PFOA-exposed liver cells in vivo and in vitro were used to discuss underlying mechanism related to PFOA-induced metabolic dysfunction. In serological tests, PFOA-exposed mice showed increased treads of liver functional enzymes in alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin (T-BIL), trypsinase, low density lipoprotein-cholesterol (LDL-C), and insulin, while blood glucose, high density lipoprotein-cholesterol (HDL-C), and glucagon levels were reduced. In histocytological observations, PFOA-exposed liver showed visible cytoplasmic vesicles, and intact pancreatic islets were observed in PFOA-exposed pancreas. Additionally, increased insulin-positive cells and reduced glucagon-positive cells were detected in PFOA-exposed islets. As shown in immunoassays, PFOA-exposed liver resulted in elevations of cluster of differentiation 36 (CD36)-labeled cells and CD36 protein. In mouse liver cell study, PFOA-exposed cells showed increased cell apoptotic count, and increased phosphorylated levels of Bcl-2 and Bad in the cells. Furthermore, PFOA-exposed liver cells exhibited elevations of CD36-labeled cells and CD36 protein. Taken together, the present data demonstrate that acute exposure to PFOA-impaired liver function is associated with inducting CD36 expression and apoptosis, as well as disrupting key hormones in the pancreas.     

Effect of smelter emissions on the hemogram of the deer mouse (Peromyscus maniculatus)

Blood samples were obtained from 284 deer mice (Peromyscus maniculatus) live-trapped near two base metal smelters in northern Manitoba, Canada. Hemoglobin and hematocrit values were significantly higher in mice trapped within 40-70 km of the smelters compared to those in control areas 185 to 190 km from the smelter. These increases appear to be normal physiological responses to a mild respiratory stress, as in a true secondary polycythemia of anoxic tupe. The mice are sensitive bioindicators of sulphur dioxide emissions that apparently induce the higher blood values.     

Effects of metabolites of benzo(a)pyrene on unschedule DNA synthesis in BALB/3T3 cell line

In order to explore the damage from metabolites of benzo(a)pyrene on DNA of mammalian cells, the effects of four metabolites of benzo(a)pyrene (anti-BPDE, syn-BPDE, 3-OH-BP and 9-OH-BP) on synthesis of DNA and unschedule DNA synthesis (UDS) in BALB/3T3 cells were assayed, by methods of single-labeling and double-labeling. The results showed that all of the four agents were able to increase the synthesis of DNA, but only three of them (apart from syn-BPDE) induced UDS in BALB/3T3 cells. The above indicates that the metabolites of benzo(a)pyrene are able to damage DNA in BALB/3T3 cells, and that this effect may be relative to the sterical structure of metabolites of benzo(a)pyrene.     

Aryl hydrocarbon receptor (AhR) inducers and estrogen receptor (ER) activities in surface sediments of Three Gorges Reservoir,China evaluated with in vitro cell bioassays

Two types of biological tests were employed for monitoring the toxicological profile of sediment cores in the Three Gorges Reservoir (TGR), China. In the present study, sediments collected in June 2010 from TGR were analyzed for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activities. The estrogenic activity was assessed using a rapid yeast estrogen bioassay, based on the expression of a green fluorescent reporter protein. Weak anti-estrogenic activity was detected in sediments from an area close to the dam of the reservoir, and weak estrogenic activities ranging from 0.3 to 1 ng 17β-estradiol (E2) equivalents (EQ) g^?1 dry weight sediment (dw) were detected in sediments from the Wanzhou to Guojiaba areas. In the upstream areas Wanzhou and Wushan, sediments demonstrated additive effects in co-administration of 1 nM E2 in the yeast test system, while sediments from the downstream Badong and Guojiaba areas showed estrogenic activities which seemed to be more than additive (synergistic activity). There was an increasing tendency in estrogenic activity from upstream of TGR to downstream, while this tendency terminated and converted into anti-estrogenic activity in the area close to the dam. The AhR activity was detected employing rat hepatoma cell line (H4IIE). EROD activities were found homogenously distributed in sediments in TGR ranging from 200 to 311 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) EQ g^?1 dw for total AhR agonists and from 45 to 76 pg TCDD EQ g^?1 dw for more persistent AhR agonists. The known AhR agonists polycyclic aromatic hydrocarbon, polychlorinated biphenyl, and PCDD/F only explained up to 8 % of the more persistent AhR agonist activity in the samples, which suggests that unidentified AhR-active compounds represented a great proportion of the TCDD EQ in sediments from TGR. These findings of estrogenic potential and dioxin-like activity in TGR sediments provide possible weight-of-evidence of potential ecotoxicological causes for the declines in fish populations which have been observed during the past decades in TGR.     

Effect of copper on soil functional stability measured by relative soil stability index (RSSI) based on two enzyme activities

Copper can affect essential processes in soils, often for long periods. Enzyme activity is considered a sensitive indicator to evaluate soil health and the potential toxic impact of a soil contaminant. Nevertheless, there is heterogeneity in the responses from enzyme activity assays because of the influence of pH and other physicochemical parameters on both enzyme activity and metal speciation. This leads to complications when comparing soils and limits the validity of the results. To overcome these problems, this paper evaluates resistance and recovery, quantified by using a relative soil stability index (RSSI), of the beta-glucosidase and protease activities towards an additional heat disturbance (17 h at 60 degrees C) in soils where soil organic matter, pH and Cu content were modified in a factorial setup. Chemical analyses (dissolved Cu, pCu(2+), dissolved organic carbon, pH) were performed both before the heat-perturbation and after the enzyme activity monitoring period. Results show that soil pH did not interfere with the RSSI scores of both enzymes. beta-glucosidase RSSI scores were scarcely affected by copper, making it inappropriate for evaluating copper-induced stress to soils. Protease activity shows stimulations of up to 2.5 times the activity of the unperturbed control in uncontaminated samples only. Thus, the protease RSSI score seems a good indicator for soil health relative to copper contamination given that all samples were affected by the presence of copper and high correlations were observed between RSSI scores and the different copper forms.     

Effect of fungicides on plant growth promoting activities of phosphate solubilizing Pseudomonasputida isolated from mustard (Brassica compestris) rhizosphere

This study was navigated to examine the effects of fungicide-stress on the activities of plant-growth-promoting rhizobacterium Pseudomonasputida with inherent phosphate solubilizing activity. The fungicide-tolerant and phosphate solubilizing P.putida strain PS9 was isolated from the mustard rhizosphere and tentatively identified following standard morphological, physiological and biochemical tests. To further consolidate the identity of the strain PS9, the 16S rDNA sequence analysis was performed. Following the BLAST program, the strain PS9 was identified as P.putida. In the presence of the varying concentrations (0-3200 μg mL⁻¹; at a two fold dilution interval) of four fungicides of different chemical families (tebuconazole, hexaconazole, metalaxyl and kitazin) amended in minimal salt agar medium, the P.putida strain PS9 showed a variable tolerance levels (1400-3200 μg mL⁻¹) against the tested fungicides. The strain PS9 produced plant-growth-promoting (PGP) substances in significant amount in the absence of fungicides. In general, fungicides applied at the recommended, two and three times of the recommended rates, decreased the PGP attributes of P.putida the strain PS9 and affected the PGP activities in concentration-dependent manner. Fungicides at the recommended dose had minor reducing effect while the doses higher than the recommended dose significantly reduced the PGP activities (phosphate solubilization, salicylic acid, 2,3-dihydroxy benzoic acid, and indole-3-acetic acid production except exo-polysaccharides, hydrogen cyanate and ammonia production). Of the four fungicides, tebuconazole generally, showed maximum toxicity to the PGP activities of the strain PS9. This study inferred that fungicides must be examined in vitro for their possible adverse effects on soil micro flora before their application in agricultural fields. Moreover, the results also suggested the prerequisite of application of fungicide-tolerant PGPR strains as bioinoculants so that their PGP activities may not be suppressed under fungicide stress.     

Effects of mono-(2-ethylhexyl) phthalate (MEHP) on chicken germ cells cultured in vitro

In recent decades, many toxicological tests based on in vivo or in vitro models, mainly from mammalian (rat–mouse) and fish species, were used to assess the risks raised by contact or ingestion of molecules of pharmaceutical, agricultural, or natural origin. But no, or few, in vitro tests using other non-mammalian models such as bird have been explored despite their advantages: the embryonic gonads of birds have a high plasticity of development sensitive to estrogen, and sperm production is nearly two times faster than in rodents. Hence, we have established an in vitro culture of germ cells and somatic cells from chicken post-natal testis, and we have evaluated the sensitivity against the endocrine disruptor compound mono-(2-ethylhexyl) phthalate (MEHP) in comparison to previous studies using rodent and human models. After 96 h of exposure in presence of 10 μM MEHP, chicken seminiferous tubules cultures present a structural alteration, a reduction in cell proliferation and in germ cells population. Apoptosis of germ and somatic cells increases in presence of 1 μM MEHP. Furthermore, MEHP does not affect inhibin B and lactate production by Sertoli cells. These results are in accordance with previous studies using rat, mice, or human culture of testicular cells and in similar range of exposures or even better sensitivity for some “end-points” (biological parameters). In conclusion, the establishment of this postnatal testicular cells culture could be considered as an alternative method to in vivo experiments frequently used for evaluating the impact on the terrestrial wildlife species. This method could be also complementary to mammal model due to the limiting number of animals used and its elevated sensitivity.