Saro Jayaraman

Characterization of the hypothalamic-pituitary-adrenal axis response to atrazine and metabolites in the female rat.

Atrazine (ATR) has recently been shown to activate the hypothalamic-pituitary-adrenal (HPA) axis in rodents. The current study investigated the effect of ATR and two of its chlorinated metabolites, desisopropylatrazine (DIA) and diamino-s-chlorotriazine (DACT), on the HPA axis in the Long-Evans female rat. A single oral gavage administration of 75 mg/kg ATR or 60.2 mg/kg DIA (a dose equimolar to the applied ATR dose) during the morning of proestrus resulted in significant, acute increases in circulating adrenocorticotropic hormone (ACTH), corticosterone, and progesterone. Oral doses of ATR or DIA were given daily over the course of the 4-day ovarian cycle starting on the day of vaginal estrus, resulted in a similar, dose-responsive activation of the HPA axis. The increase in ACTH, corticosterone, and progesterone by these compounds was of a similar magnitude to that produced by 5-min restraint stress. Single or multiple oral exposures to DACT, on the other hand, did not significantly alter pituitary-adrenal hormone release. These results were observed despite plasma levels of DACT being higher than any other metabolite at the time of hormone measurement. Overall, circulating metabolite concentrations following equimolar dosing were much higher than those observed after ATR administration. Additional studies indicated that the activation of the HPA axis by oral exposure to ATR and DIA was not due simply to the stimulation of gastrointestinal afferents. Similar responses were observed in rats which received an oral dose of ATR following bilateral subdiaphramatic vagotomy and following intravenous administration of DIA in jugular vein catheterized animals. We conclude that ATR and the metabolite DIA significantly activate the HPA axis following oral exposure in the female rat. Activation of this endocrine axis by these chlorotriazines could contribute to the induced changes of female reproductive function reported previously.

Extraction of organic contaminants from marine sediments and tissues using microwave energy

In this study, we compared microwave solvent extraction (MSE) to conventional methods for extracting organic contaminants from marine sediments and tissues with high and varying moisture content. The organic contaminants measured were polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, and polycyclic aromatic hydrocarbons (PAHs). Initial experiments were conducted on dry standard reference materials (SRMs) and field collected marine sediments. Moisture content in samples greatly influenced the recovery of the analytes of interest. When wet sediments were included in a sample batch, low recoveries were often encountered in other samples in the batch, including the dry SRM. Experiments were conducted to test the effect of standardizing the moisture content in all samples in a batch prior to extraction. SRM1941a (marine sediment). SRM1974a (mussel tissue), as well as QA96SED6 (marine sediment), and QA96TIS7 (marine tissue), both from 1996 NIST Intercalibration Exercise were extracted using microwave and conventional methods. Moisture levels were adjusted in SRMs to match those of marine sediment and tissue samples before microwave extraction. The results demonstrated that it is crucial to standardize the moisture content in all samples, including dry reference material to ensure good recovery of organic contaminants. MSE yielded equivalent or superior recoveries compared to conventional methods for the majority of the compounds evaluated. The advantages of MSE over conventional methods are reduced solvent usage, higher sample throughput and the elimination of halogenated solvent usage.

Chlorotriazine Herbicides and Metabolites Activate an ACTH-dependent Release of Corticosterone in Male Wistar Rats

Previously, we reported that atrazine (ATR) alters steroidogenesis in male Wistar rats resulting in elevated serum corticosterone (CORT), progesterone, and estrogens. The increase in CORT indicated that this chlorotriazine herbicide may alter the hypothalamic-pituitary-adrenal axis. This study characterizes the temporal changes in adrenocorticotropic hormone (ACTH), CORT, and P4 in male Wistar rats following a single dose of ATR (0, 5, 50, 100, and 200 mg/kg), simazine (SIM; 188 mg/kg), propazine (PRO; 213 mg/kg), or primary metabolites, deisopropylatrazine (DIA; 4, 10, 40, 80, and 160 mg/kg), deethylatrazine (DEA; 173 mg/kg), and diamino-s-chlorotriazine (DACT; 3.37, 33.7, 67.5, and 135 mg/kg). The maximum dose for each chemical was the molar equivalent of ATR (200 mg/kg). Significant increases in plasma ACTH were observed within 15 min, following exposure to ATR, SIM, PRO, DIA, or DEA. Dose-dependent elevations in CORT and progesterone were also observed at 15 and 30 min post-dosing with these compounds indicating an activation of adrenal steroidogenesis. Measurement of the plasma concentrations of the parent compounds and metabolites confirmed that ATR, SIM, and PRO are rapidly metabolized to DACT. Although DACT had only minimal effects on ACTH and steroid release, dosing with this metabolite resulted in plasma DACT concentrations that were 60-fold greater than that observed following an equimolar dose of ATR and eightfold greater than equimolar doses of DIA or DEA, indicating that DACT is not likely the primary inducer of ACTH release. Thus, the rapid release of ACTH and subsequent activation of adrenal steroidogenesis following a single exposure to ATR, SIM, PRO, DIA, or DEA may reflect chlorotriazine-induced changes at the level of the brain and/or pituitary.

Evolution of tolerance to PCBs and susceptibility to a bacterial pathogen (Vibrio harveyi) in Atlantic killifish (Fundulus heteroclitus) from New Bedford (MA, USA) harbor.

A population of the non-migratory estuarine fish Fundulus heteroclitus (Atlantic killifish) resident to New Bedford (NB), Massachusetts, USA, an urban harbor highly contaminated with polychlorinated biphenyls (PCBs), demonstrates recently evolved tolerance to some aspects of PCB toxicity. PCB toxicology, ecological theory, and some precedence supported expectations of increased susceptibility to pathogens in NB killifish. However, laboratory bacterial challenges of the marine pathogen Vibrio harveyi to wild fish throughout the reproductive season and to their mature laboratory-raised progeny demonstrated comparable survival by NB and reference killifish, and improved survival by NB males. These results are inconsistent with hypothesized trade-offs of adaptation, and suggest that evolved tolerance in NB killifish may include mechanisms that minimize the immunosuppressive effects of PCBs. Compensatory strategies of populations persisting in highly contaminated environments provide a unique perspective for understanding the long-term ecological effects of toxic chemicals.

Environmental stress and recovery: the geochemical record of human disturbance in New Bedford Harbor and Apponagansett Bay, Massachusetts (USA)

Sediments record the history of contamination to estuaries. Analysis of the concentrations of toxic organic compounds, contaminant and crustal metals, organic carbon content and isotopic composition in sediment cores from two estuarine systems in Buzzards Bay allowed reconstruction of human impacts over 350 years. Vertical distributions of the contaminants correlate with changes in the nature of watershed/estuarine activities. All contaminants were highly enriched (tens to hundreds times background) in modern New Bedford Harbor sediments. Enrichment began around the turn of the 20th century for all but PCBs, which were first synthesized in the 1930s. An increase in organic carbon content and a shift of carbon isotopes toward a more terrestrial signature illustrates increasing anthropogenic impact in New Bedford as population grew along with the industrial base. Institution of environmental protection measures in the late 20th century was reflected in decreased, although still substantially elevated, concentrations of contaminants. A lack of industrial development in Apponagansett Bay resulted in much lower concentrations of the same indicators, although specific contaminants related to the early whaling industry increased significantly above background as early as the late 18th century. The similarity of indicators in older portions of cores from NBH and unimpacted Apponagansett Bay demonstrates that cores can be used to establish reference conditions as successfully as using separate sites judged a priori to represent the reference state. The historical reconstruction approach provides the basis for establishing relationships between environmental stressors and factors that drive the stressors, as well as a framework for the assessment of ecological response(s) to environmental stressors over a range of time and/or exposure scales.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

PCBs and DDE in Tree Swallow (Tachycineta bicolor) Eggs and Nestlings from an Estuarine PCB Superfund Site, New Bedford Harbor, MA, U.S.A.

While breeding tree swallows (Tachycineta bicolor) have been used as biomonitors for freshwater sites, we report the first use of this species to assess contaminant bioaccumulation from estuarine breeding grounds into these aerial insectivores. Eggs and nestlings were collected from nest boxes in a polychlorinated biphenyl (PCB) contaminated estuary, the New Bedford Harbor Superfund site (NBH, Massachusetts, USA), and a reference salt marsh, Fox Hill (FH, Jamestown, Rhode Island, USA). Sediments, eggs, and nestlings were compared on a ng g(-1) wet weight basis for total PCBs and DDE (1,1-bis-(4-chlorophenyl)-2,2-dichloroethene), metabolite of DDT (1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane). NBH samples contained high concentrations of PCBs compared to FH for sediment (36,500 and 0.2), eggs (11,200 and 323), and nestlings (16,800 and 26). PCB homologue patterns linked tree swallow contamination to NBH sediment. NBH samples were also contaminated with DDE compared to FH for sediment (207 and 0.9) and nestlings (235 and 30) but not for eggs (526 and 488), suggesting both NBH and nonbreeding ground sources for DDE. The relationships between sediment and tree swallow egg and nestling PCBs were similar to those reported for freshwater sites. Like some highly contaminated freshwater sites, NBH PCB bioaccumulation had little apparent effect on reproductive success.

Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat.

Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and testis (intact) proteomes in rats after 3 days of exposure. The adrenal accounted for most of the serum progesterone and all of the corticosterone increases in intact and castrated males. Serum luteinizing hormone, androstenedione, and testosterone in intact males shared a non-monotonic response suggesting transition from an acute stimulatory to a latent inhibitory response to exposure. Eight adrenal proteins were significantly altered with dose. There were unique proteomic changes between the adrenals of intact and castrated males. Six testis proteins in intact males had non-monotonic responses that significantly correlated with serum testosterone. Different dose-response curves for steroids and proteins in the adrenal and testis reveal novel adverse outcome pathways in intact and castrated male rats.

Expression and function of ryanodine receptor related pathways in PCB tolerant Atlantic killifish (Fundulus heteroclitus) from New Bedford Harbor, MA, USA.

Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic effects through the aryl hydrocarbon receptor (AhR) are well studied. In NBH, non-dioxin like PCBs (NDL PCBs), which lack activity toward the AhR, vastly exceed levels of DL congeners yet how killifish counter NDL toxic effects has not been explored. In mammals and fish, NDL PCBs are potent activators of ryanodine receptors (RyR), Ca(2+) release channels necessary for a vast array of physiological processes. In the current study we compared the expression and function of RyR related pathways in NBH killifish with killifish from the reference site at Scorton Creek (SC, MA). Relative to the SC fish, adults from NBH displayed increased levels of skeletal muscle RyR1 protein, and increased levels of FK506-binding protein 12 kDa (FKBP12) an accessory protein essential for NDL PCB-triggered changes in RyR channel function. In accordance with increased RyR1 levels, NBH killifish displayed increased maximal ligand binding, increased maximal response to Ca(2+) activation and increased maximal response to activation by the NDL PCB congener PCB 95. Compared to SC, NBH embryos and larvae had increased levels of mtor and ryr2 transcripts at multiple stages of development, and generations, while levels of serca2 were decreased at 9 days post-fertilization in the F1 and F2 generations. These findings suggest that there are compensatory and heritable changes in RyR mediated Ca(2+) signaling proteins or potential signaling partners in NBH killifish.

High performance liquid chromatographic analysis of phytoplankton pigments using a C16-amide column.

In this study, a reverse-phase HPLC method incorporating a ternary solvent system was developed to analyze most polar and non-polar chlorophylls and carotenoids present in phytoplankton. The method is based on an RP-C₁₆-Amide column and provided excellent peak resolution of most taxonomically important pigments and an elution profile different than C₈ or C₁₈ columns provide. Analysis of mixed pigment standards, extracts of phytoplankton monocultures, and field samples showed that this method was able to resolve more than sixty pigments, ranging from very polar acidic chlorophylls to the non-polar hydrocarbon carotenes in less than 36 min. This included chlorophylls c₁, c₂ and c₃, divinyl chlorophylls a and b, the carotenoids lutein and zeaxanthin and some recently discovered pigments. The ability of this method to resolve divinyl chl b from monovinyl chl b and divinyl chl a from monovinyl chl a is particularly important for the quantification and identification of the marine cyanobacteria Prochlorococcus spp. in oceanic waters. The described protocol is sensitive and reproducible and can be used to assess the distribution and dynamics of major phytoplankton groups in marine and freshwater ecosystems.