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DIFFERENTIAL EFFECTS OF TWO LOTS OF AROCLOR 1254R: CONGENER ANALYSIS AND NEUROCHEMICAL ENDPOINTS.
Citation:
Kodavanti, P., N. Kannan, N. Yamashita, E. C. DerrYellin, T. R. Ward, D. E. Burgin, H A. Tilson, AND L S. Birnbaum. DIFFERENTIAL EFFECTS OF TWO LOTS OF AROCLOR 1254R: CONGENER ANALYSIS AND NEUROCHEMICAL ENDPOINTS. Presented at 20th Intl Symposium on Halogenated Environmental Organic Pollutants & POPs), Monterey, CA, August 13-17, 2000.
Description:
Introduction
Polychlorinated biphenyls (PCBs) are widely used in industry as heat transfer and dielectric fluids for transformers and capacitors1. PCBs were commercially produced as AroclorR mixtures in USA by the chlorination of biphenyl. Although all 209 congeners can be synthesized, the reaction conditions favor certain substitution reactions leading to particular composition of the technical mixtures, which are identified by the weight percentage of chlorine content. For example, Aroclor 1254 contains 54% chlorine by weight, as indicated by the last two digits in the numerical designation 1-2.
PCBs cause a wide range of effects in humans including chloracne, diverse hepatic effects, decreased birth weight in the offsprings, decreased pulmonary function, subtle endocrine disturbances, cancer, and learning and memory deficits3-7. Several of these effects have been demonstrated in animals following exposure to commercial PCB mixtures such as Aroclor 12543-5. This information has been used in the risk/exposure assessment of PCBs and related chemicals1,5. However, recent reports indicate substantial differences in the congener composition among Aroclor lots6, 7. Toxicity studies with Aroclor 1254 have given varied results8, 9, but these differences were attributed to the animal species or dosing rather than to the chemical composition. The current study was designed to study the effects of two lots of Aroclor 1254 (Lot #s 6024 and 124-191) on the neurochemical endpoints in vitro and compare the effects with their PCB congener composition and other contaminants.
Materials and Methods
Chemicals. Aroclor 1254 (>99% purity) with lot numbers 6024 and 124-191 were purchased from AccuStandard, New Haven, CT. For neurochemical experiments, stock solutions were prepared in dimethyl sulfoxide (DMSO). DMSO alone (2 ml/ml) had no significant effect on the endpoints.
Congener-Specific Analysis of PCBs, Dioxins (PCDDs), Furans (PCDFs), and Napthalenes (PCNs) in Two Lot s of Aroclor 1254. Congener-specific analysis of PCBs was performed according to previously established method10. Non-ortho PCBs were separated from other dominant ortho-PCBs for their trace level determination using Cosmosil 5-PYE column-HPLC method and analyzed in high resolution multidimensional gas chromatography-electron capture detector (HRMDGC-ECD). The PCDD, PCDF, and PCN analyses were performed as per the methods described previously11, 12.
Neurochemical endpoints. Adult rat cerebella were fractionated to obtain microsomes and mitochondria13. Intracellular Ca2+ buffering was determined by measuring the uptake of 45Ca2+ by microsomes and mitochondria14. Granule cells from cerebellum of 7 day old rats were isolated by the enzymatic disruption of cells15. These cells were maintained for 7 days in vitro in culture and used for protein kinase C (PKC) translocation studies, determined by measuring 3H-phorbol ester binding16.
Statistics. The neurochemical data were analyzed by Two-Way Analysis of Variance (ANOVA) followed by Dunnett=s test. The IC50 (concentration that inhibits control activity by 50%) and E50 (concentration that increases control activity by 50%) values for 45Ca2+ -uptake and 3H-phorbol ester binding, respectively, were calculated from the regression line fit to the linear portion of the curve and were compared using student=s t-test between two lots. The significance was set at p < 0.05.
Results and Discussion
Results from this study indicate that the composition of PCB congeners were significantly different between the two lots of Aroclor 1254 (Table 1). Dioxins were not detected in either lot (< 2 parts per billion), as anticipated. PCNs were similar in both lots. However, PCDFs were detectable and Lot # 6024 has 3.4 times more than Lot # 124-191 (data not shown). Differences in the congener composition among different lots of Aroclor mixtures have been previously reported6, 7. However, this is the first detailed report about the congener-specific analysis along with the contaminants of these two lots of Aroclor 1254 (Source: AccuStandard, Inc), a widely used commercial mixture in USA for conducting scientific research. Of the two lots of Aroclor 1254 tested, Lot # 6024 has more non-ortho PCBs and PCDFs contributing to higher TEQ values. Recently, Frame7 discussed the manufacturing process of several Aroclors. Lot # 124-191 of Aroclor 1254 has the typical PCB congener distribution. Lot #6024 has been traced back to Monsanto Lot # KI-6024 and represents the late (1974-1976) production which used a two-stage chlorination procedure resulting in greatly increased levels of non-ortho, mono-ortho congeners and PCDFs with high TEF values7.
The biological activity of two lots of Aroclor 1254 was tested on two previously established neurochemical endpoints such as intracellular Ca2+ buffering and PKC translocation17. Intracellular Ca2+ buffering is essential in the maintenance of normal calcium homeostasis. When intracellular free Ca2+ levels increase, PKC may translocate from cytosol to the membrane where it gets activated. PCBs have been shown to increase intracellular free Ca2+ in a number of cell systems15, 18. When these two lots of Aroclors were tested, the effects were significantly different (Table 2). Intracellular Ca2+ buffering by microsomes and mitochondria was significantly inhibited by both lots of Aroclor 1254 in a concentration-dependent manner. Lot # 6024 seems to be more potent compared to Lot # 124-191. This difference in the potency increased several fold when the concentration is transformed to TEQ values demonstrating that the greater effect with Lot # 6024 is not due to the greater Ah-receptor binding activity alone. PKC translocation measured as 3H-phorbol ester binding was significantly increased by both lots of Aroclor 1254; Lot # 124-191 was significantly more active than Lot # 6024. As seen with Ca2+ buffering, the difference in the potency also increased several fold when the concentration is transformed to TEQ (ng/ml) suggesting that the dioxin-like congeners are not responsible for this effect. The differential effects of two lots of Aroclor 1254 on the selected neurochemical endpoints were not explained by the differences in their TEQs. However, other endpoints such as hepatic induction of EROD and MROD were explained by their TEQs19. These results suggest that overall toxicity of complex mixtures can not be entirely predicted based on the TEQ values and caution should be used when making risk assessment decisions about chemical mixtures which involve both Ah-receptor-dependent and -independent mechanisms.