The Effects of Developmental Exposure to Aroclor 1254Ò on Serotonin Concentrations in Black Swiss Webster Mice
Polychlorinated biphenyls (PCBs) are chemically, thermally, biologically stable compounds that were previously used in multiple industries and released into the environment (Mariussen, 2001). The 209 possible PCB congeners are manually synthesized by chlorine substitution on biphenyl rings. The production of PCBs was banned in most industrialized countries since the 1970s, and has been banned in the United States since 1977. Improper disposal of these compounds, and the popular use of them for decades, has caused persistent damage to ecosystems because of contaminated soil and water supplies (Jacobson, 1984). Research investigating the physiological impact of PCB exposure has shown endocrine disruption (Mariussen, 2001).
Exposure to PCBs in mothers is particularly harmful due to the lipophilic nature of the chemicals (Jacobson et al, 1984). Exposure to PCBs prior to pregnancy leads to storage in fatty tissue and later incorporation into breast milk composition (Jacobson et al., 1984). The appearance of PCBs in breast milk increases the risk of endocrine disruption in offspring exposed during gestation and lactation. However, the extent of the endocrine disruption in animals exposed during development requires more investigation.
Venkataraman et al., found that PCBs reduce the production of melatonin in adult animals, causing oxidative stress in brain regions that melatonin would normally protect. The activity of N-acetyltransferase (NAT), the rate-limiting enzyme during melatonin production, is inversely affected by PCB exposure in animals exposed during development (Hirschey et al., 2007). Melatonin is part of the hormonal cycle that regulates circadian rhythm and is initially produced during scotal phase by the acylation of serotonin by the enzyme NAT forming N-acetylserotonin. Melatonin is then produced by the conversion of N-acetylserotonin by the enzyme hydroxyindole O-methyltransferase (Arendt, 2000). A reduction in serotonin within this system could cause a cascade affect resulting in less than optimal melatonin production. This would be detrimental to the regulation of the endocrine system, sleep wake cycles, and circadian rhythm.
In this proposed study we will investigate the effect that the PCB mixture Aroclor 1254® has on the production of serotonin in mice exposed during development. Developmental exposure to PCBs has an effect on NAT activity in mice, but it is not known if this is caused by a direct impact on NAT production, or a decrease in the conversion of tryptophan to serotonin. We will investigate the concentration of serotonin in the brain and blood of mice after gestational and lactational exposure to Arclor 1254® in two concentrations (1.25 ppm and 12.5 ppm). Although 90% of the serotonin found in the body is produced primarily in enterochromaffin cells are found in the duodenum of the small intestine, most of this serotonin is released into the blood (Doe-Young & Camilleri, 2000; Bertaccini, 1960; Espramer & Testini, 1959). By testing the blood we will be indirectly measuring the production of serotonin in the enterochromaffin cells of the small intestine. The remaining 10% of serotonin is found within the brain. We hypothesize that as the concentration of PCB is increased the production of serotonin will decrease in both areas. We predict that this inverse relationship will develop in a dose dependent response in the treatment groups that receive 1.25ppm and 12.5ppm PCBs.
Materials and Methods
A total of 24 female and 10 male Black Swiss-Webster mice will be introduced to the lab facility and acclimated for a minimum of 7 days. They will be housed in a climate-controlled room (20-22°C), with white noise played continuously to prevent acclimation to silence. The room will be on a 12L-12D reversed light cycle. If research should need to take place during the dark cycle, then red light will be used to observe the animals without disrupting this cycle. Each mouse will be housed singly in a standard cage (30.5cm x 23cm x 15.25 cm h) containing approximately two inches of wood shavings. Standard ground rodent chow containing 14% protein and tap water will be provided ad libitum.
During breeding a female mouse will be housed in a male’s cage for 5 days, to insure fertilization. Conception will be confirmed with a weight gain of 1g. After the five-day breeding period, the pairs will be separated into single cages. Each female will be weighed daily to track pregnancy progress. Litters will be culled 3-days postnatally to 4 males and 4 females when possible.
Treatments
As females are separated into home cages they will be assigned to one of three treatments; including 1.25 ppm PCBs, 12.5 ppm PCBs, or control. One treatment group of 8 pregnant females will be provided with standard ground rodent chow containing no PCBs. A second treatment group of 8 pregnant females will be provided with 1.25ppm A1254 mixed into ground rodent chow (1.25mg/kg food). A third treatment group of 8 pregnant females will be given 12.5 ppm A1254 mixed into ground rodent chow (12.5mg/kg food). Food remaining in dishes will be weighed along with any food sifted from cage shavings to calculate food consumption and PCBs dose per gram of body weight.
At PND 15, 2 females and 2 males from each litter (0 PCB, 1.25 ppm PCB, and 12.5 ppm PCB) will be injected interperitonally with 150 mg/kg bodyweight of 5-ethyl-5(1-mthylbutyl)-2,4,6-trihexahydropyrimidine, Nembutal, supplied by Sigma-Aldrich followed by quick decapitation. Brains will be dissected free and quickly frozen for analysis of serotonin and PCB concentration. Trunk blood will be collected in glass conical tubes and allowed to clot. Blood will be centrifuged for 15 min and serum will be frozen for analysis of serotonin. The remaining pups, 2 females and 2 males per litter, will be euthanized at PND 30 and tissue samples and blood frozen for analysis of serotonin and PCB concentration.
Tissue Preparation and Serotonin & PCB Determination
The brain tissue samples will be thawed then homogenized in 1M NASO4 buffer prior to being subjected to the serotonin and PCB assays. Serotonin levels in the brain and blood will be measured using a commercially available Serotonin Radioimmunoassay (RIA) kit from MP Biomedical, Solon, OH. PCB levels will be measured in the brain and blood using a commercially available PCB assay kit from Abraxis, Warminster, PA.
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