Evaluation of the immunomodulatory effects of C9-13-CPs in macrophages.

Short-chain chlorinated paraffins (SCCPs) have been listed as a new class of persistent organic pollutants by the Stockholm Convention. SCCPs exhibit carcinogenic-, endocrine-, and metabolism-disrupting effects. However, the knowledge of the immunomodulatory effects of SCCPs and their underlying mechanisms, especially in specific immune cells, remains limited. In addition to SCCPs, C9-13-CPs have also been detected in humans. In this study, murine RAW264.7 macrophages were exposed to C9-13-CPs at environmentally relevant concentrations to investigate whether or how C9-13-CPs exhibit immunomodulatory effects. The results showed that the exposure of RAW264.7 cells to C9-13-CPs increased cell viability, as assayed by MTT analysis at 490 nm, and also promoted cell proliferation, as indicated by EdU uptake assay, which was measured at excitation and emission wavelengths of 488 and 512 nm, respectively. In addition, exposure to C9-13-CPs not only led to elevated ATP level and intracellular Ca2+ level but also caused AMPK signaling activation and NF-κB signaling inhibition. Moreover, molecular docking showed that the ß2-AR receptor could bind to C9-13-CPs. Taken together, these results suggest that the immune dysfunction of RAW264.7 cells caused by C9-13-CPs is closely related to the ß2-AR/AMPK/NF-κB signaling axis.

Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1.

In the development of the cellular slime mold Dictyostelium discoideum, the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH(2); 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH(2)-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH(2)-conjugated resin, and this binding was inhibited by co-addition of 20 microM DIF-1 or DIF-1-NH(2). In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in approximately 60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function.

Immune cell counts and risks of respiratory infections among infants exposed pre- and postnatally to organochlorine compounds: a prospective study.

BACKGROUND: Early-life chemical exposure may influence immune system development, subsequently affecting child health. We investigated immunomodulatory potentials of polychlorinated biphenyls (PCBs) and p,p'-DDE in infants. METHODS: Prenatal exposure to PCBs and p,p'-DDE was estimated from maternal serum concentrations during pregnancy. Postnatal exposure was calculated from concentrations of the compounds in mother's milk, total number of nursing days, and percentage of full nursing each week during the 3 month nursing period. Number and types of infections among infants were registered by the mothers (N = 190). White blood cell counts (N = 86) and lymphocyte subsets (N = 52) were analyzed in a subgroup of infants at 3 months of age. RESULTS: Infants with the highest prenatal exposure to PCB congeners CB-28, CB-52 and CB-101 had an increased risk of respiratory infection during the study period. In contrast, the infection odds ratios (ORs) were highest among infants with the lowest prenatal mono-ortho PCB (CB-105, CB-118, CB-156, CB-167) and di-ortho PCB (CB-138, CB-153, CB-180) exposure, and postnatal mono- and di-ortho PCB, and p,p'-DDE exposure. Similar results were found for pre- and postnatal CB-153 exposure, a good marker for total PCB exposure. Altogether, a negative relationship was indicated between infections and total organochlorine compound exposure during the whole pre- and postnatal period. Prenatal exposure to CB-28, CB-52 and CB-101 was positively associated with numbers of lymphocytes and monocytes in infants 3 months after delivery. Prenatal exposure to p,p'-DDE was negatively associated with the percentage of eosinophils. No significant associations were found between PCB and p,p'-DDE exposure and numbers/percentages of lymphocyte subsets, after adjustment for potential confounders. CONCLUSION: This hypothesis generating study suggests that background exposure to PCBs and p,p'-DDE early in life modulate immune system development. Strong correlations between mono- and di-ortho PCBs, and p,p'-DDE exposures make it difficult to identify the most important contributor to the suggested immunomodulation, and to separate effects due to pre- and postnatal exposure. The suggested PCB and p,p'-DDE modulation of infection risks may have consequences for the health development during childhood, since respiratory infections early in life may be risk factors for asthma and middle ear infections.

Diagnostic relevance of the determination of lymphocyte subpopulations in environmental medicine.

Earlier hopes that determination of lymphocyte subpopulations might become a strong diagnostic tool in environmental medicine have not been fulfilled in recent years. Analysis of the scientific literature rather shows that there are only few examples for environmental exposures causing reproducible shifts of lymphocyte subpopulations. Moreover, current knowledge suggests that "environmental diseases" are not associated with characteristic changes of subpopulation patterns. If lymphocyte subpopulations are analyzed, each diagnostic step, including indication, sample handling, analytic procedure and data-interpretation, should adhere to good quality criteria. Taking all together, the determination of lymphocyte subpopulations in the context of environmental medicine comes under category IV of the criteria of the Commission for Methods and Quality Assurance in Environmental Medicine of the German federal health authority (Robert Koch-Institute; RKI): "A procedure cannot be recommended because there is not sufficient information to justify it" (here: no solid trends in epidemiological examinations), "and because theoretical considerations speak against an application" (here: high physiological variability and missing exposure or substance specificity).

Generation of antibodies to Di- and trichloroacetylated proteins and immunochemical detection of protein adducts in rats treated with perchloroethene.

Antibodies directed against chemical specific protein modifications are valuable tools to detect and comparatively quantify protein modifications. Both Nepsilon-(dichloroacetyl)-L-lysine and Nepsilon-(trichloroacety)l-L-lysine have been detected as modified amino acids in liver and kidneys of rats treated with perchloroethene (PER) after proteolysis. These protein modifications are formed by the interaction of reactive metabolites formed from PER with proteins. In this study we developed monospecific antibodies to dichloroacetylated and to trichloroacetylated amino acids to detect modified proteins in the target organs of PER toxicity. These antibodies were prepared by immunization of rabbits with modified keyhole limpet hemocyanin (KLH) coupled with either the dichloroacetyl or trichloroacetyl moiety. Enzyme-linked immunosorbent assays (ELISA) indicated that the polyclonal rabbit sera recognized dichloroacetylated or trichloroacetylated rabbit serum albumin (RSA), but not unmodified protein. Therefore, we further purified rabbit antisera on either Nepsilon-(dichloroacetyl)-L-lysine or Nepsilon-(trichloroacetyl)-L-lysine immobilized to immunoaffinity columns to obtain monospecific antibodies. The potential of these antibodies in the detection of di- and trichloroacetylated proteins and their selectivity for the desired dichloroacetyl or trichloroacetyl group was demonstrated in competitive enzme-linked immunosorbent assays with several structurally related compounds. Anti-dichloroacetyl (anti-DCA) antibody binding to dichloroacetylated RSA was inhibited by Nepsilon-(dichloroacetyl)-L-lysine with an IC50 value of 150 microM whereas inhibition by Nepsilon-(monochloroacetyl)-L-lysine and Nepsilon-(trichloroacetyl)-L-lysine showed an IC50 value of 100 mM. The binding of the anti-trichloroacetyl (anti-TCA) antibody to trichloroacetylated RSA was inhibited by Nepsilon-(dichloroacetyl)-L-lysine with an IC50 value of 80 mM. The inhibition by Nepsilon-(trichloroacetyl)-L-lysine was again 3 orders of magnitude stronger resulting in an IC50 value of 90 microM. Nepsilon-(acetyl)-L-lysine and unmodified RSA did not effect antibody binding to the chemically modified antigen. The antibodies were also successfully applied to detect modified proteins in subcellular fractions of liver and kidney from PER treated rats demonstrated in immunoblot. Protein adduct formation from different PER metabolism pathways was confirmed by the observation that the majority of dichloroacetylated proteins were located in kidney mitochondria and trichloroacetylated proteins were located in liver microsomes.