Responses of CYP450 dependent system to aliphatic and aromatic hydrocarbons body burden in transplanted mussels from South coast of Portugal.

Mussels Mytilus galloprovincialis were cross-transplanted at South Portugal from a reference site (site 1) to a site more contaminated with hydrocarbon compounds (site 2), and vice versa, in an active biomonitoring (ABM) concept, to assess the biotransformation capacity catalyzed by the mixed function oxygenase (MFO) system. Total alkanes (TAlk), the unresolved complex mixture (UCM), and total polycyclic aromatic hydrocarbons (TPAHs) concentration increased respectively 6, 4.4 and 4.2 fold relatively to control, in mussels transplanted from site 1 to 2. In the cross-transplant, a 48, 57 and 62% depuration of TAlk, UCM and TPAHs concentrations occurred by the end of the 3-4th week. Petrogenic and biogenic (marine and terrigenous) sources of AHs, and petrogenic and pyrolitic (biomass and oil/fuel incomplete combustion) sources of PAHs were detected at both sites. CYP450, CYT b (5) and NADPH-RED in mussels transplanted from site 1 to 2 were induced from day 0 to 28, with a total increase of 35, 32 and 35%, respectively, while biochemical equilibrium to lesser environmental contamination occurs in mussels transplanted from site 2 to 1. A significant relationship between CYP450 and NADPH-RED was found with TPAH, with distinctive behavior at the two sites. MFO system components increase with exposure time at one site and decreases in the other, reflecting an adaptation to distinct environmental hydrocarbon loads. The ABM strategy proved to be useful to understand the environment real impact on the biochemical responses in mussels' local populations. In this study, CYP450 and NADPH-RED are a useful biomarker for hydrocarbon exposure.

Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction.

Understanding the toxic mechanisms by which organisms cope to environmental stressful conditions is a fundamental question for ecotoxicology. In this study, we evaluated biochemical responses and hydrocarbons bioaccumulation of the mangrove oyster Crassostrea brasiliana exposed for 96 h to four sublethal concentrations of diesel fuel water-accommodated fraction (WAF). For that purpose, enzymatic activities (SOD, CAT, GPx, GR, G6PDH, GST and GGT), HSP60 and HSP90 immunocontent and lipid peroxidation (LPO) levels were determined in the gill and digestive gland of oysters and related to the hydrocarbons accumulated in the whole soft tissues. The results of this study revealed clear biochemical responses to diesel fuel WAF exposure in both tissues of the oyster. The capacity of C. brasiliana to bioaccumulate aliphatic and aromatic hydrocarbons in a dose-dependent manner is a strong indication of its suitability as a model in biomonitoring programs along the Brazilian coast, which was also validated by the response of the antioxidant defenses, phase II biotransformation and chaperones. HSP60 levels and GGT activity were the most promising biomarkers in the gill, while GST and GR activities stood out as suitable biomarkers for the detection of diesel toxicity in the digestive gland. The decrease of SOD activity and HSP90 levels may also reflect a negative effect of diesel exposure regardless the tissue. The present results provide a sound preliminary report on the biochemical responses of C. brasiliana challenged with a petroleum by-product and should be carefully considered for use in the monitoring of oil and gas activities in Brazil.

Effect of the water-soluble fraction of petroleum on microsomal lipid metabolism of Macrobrachium borellii (Arthropoda: Crustacea).

The effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism was studied at critical metabolic points, namely fatty acid activation, enzymes of triacylglycerol and phospholipid synthesis, and membrane (lipid packing) properties in the freshwater prawn Macrobrachium borellii. To determine the effect of the contaminant, adults and embryos at different stages of development were exposed to a sublethal concentration of WSF for 7 days. After exposure, microsomal palmitoyl-CoA synthetase (ACS) showed a two-fold increase in adult midgut gland. Embryo's ACS activity was also affected, the increment being correlated with the developing stage. Endoplasmic reticulum acylglycerol synthesis was also increased by WSF exposure in adults and stage 5 embryos, but not at earlier stages of development. Triacylglycerol synthesis was particularly increased (18.5%) in adult midgut gland. The microsomal membrane properties were studied by fluorescent steady-state anisotropy, using the rotational behavior of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Microsomes from midgut gland of WSF-exposed prawn showed no differences in fluidity. Nevertheless, microsomes incubated with WSF in vitro increased their fluidity in a temperature- and WSF concentration-dependent fashion. Both, aliphatic and aromatic hydrocarbons individually tested elicited an increase in membrane fluidity at 10 mg/l, but at 4 mg/l only nC10-C16 aliphatics did. In vivo results indicate that WSF increased the activity of microsomal enzymes that are critical in lipid metabolism, though this change was not due to direct alterations in membrane fluidity, suggesting a synthesis induction, or an enzyme-regulatory mechanism. Nevertheless, hydrocarbons elicited membrane fluidity alterations in in vitro experiments at concentrations that could be found in the environment after an oil spill.

Evaluation of the potential developmental toxicity of cyclododecatriene (CDDT).

The potential maternal and developmental toxicity of cyclododecatriene (CDDT) was assessed in rats. Groups of 22 time-mated female Crl:CD (SD) BR rats were exposed by inhalation (whole-body, 6 h/day) to either 0 (control), 10, 25, or 67 ppm CDDT over days 6-20 of gestation (days 6-20 G); the day of copulation plug detection was designated day 0 G. The dams were euthanized on day 21 G, and their abdominal and thoracic viscera were examined grossly. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal alterations. Evidence of maternal toxicity was seen at 25 and 67 ppm. There were compound-related reductions in maternal body weight and food consumption parameters as well as increased occurrences of wet and stained fur at these exposure levels. Developmental toxicity evident as reduced mean fetal weight and delayed skeletal ossification was seen only at 67 ppm. There was no evidence of either maternal or developmental toxicity at 10 ppm. Thus, the no-observed-effect level (NOEL) for maternal toxicity was 10 ppm, and the NOEL for developmental toxicity was 25 ppm. Because developmental toxicity was observed only after exposures that also produced signs of maternal toxicity, CDDT was not considered to be a selective developmental toxicant in the rat.

Transcellular signalling pathways and TNF-alpha release involved in formation of reactive oxygen species in rat alveolar macrophages exposed to tert-butylcyclohexane.

In the present work, the effects of aliphatic ( n-nonane and n-decane), alicyclic (1,2,4-trimethylcyclohexane and tert-butylcyclohexane, t-BCH) and aromatic (trimethylbenzene and tert-butylbenzene) hydrocarbon solvents on formation of reactive oxygen species (ROS) and the proinflammatory cytokine TNF-alpha in rat alveolar macrophages (AM) have been investigated. Formation of ROS was assessed by monitoring oxidation of 2',7'-dichlorofluorescin to 2',7'-dichlorofluorescein (DCF), and the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) was detected using an enzyme-linked immunosorbent assay. DCF fluorescence was elevated in a concentration-dependent manner by the alicyclic hydrocarbons. The involvement of transcellular signalling pathways in the production of ROS by t-BCH, the most active compound, was elucidated by use of specific inhibitors. Preincubation of the AM with the mitogen-activated protein kinase (ERK 1/2) inhibitor U0126, the protein kinase C inhibitor bisindolylmaleimide, the superoxide dismutase inhibitor diethyldithiocarbamate, and the iron ion chelating agent deferoxamine reduced the DCF fluorescence significantly. t-BCH gave an increase in TNF-alpha release. Further, nitric oxide production measured by a modified Griess method, and intracellular calcium concentration measured by fura-2, were increased in the rat AM after exposure to t-BCH.

Reproductive and repeated dose toxicity of cyclododecatriene (CDDT) in rats following oral (gavage) treatment.

Cyclododecatriene (CDDT, CAS No. 4904-61-4) was administered daily by oral gavage to groups of Crl:CD (SD)IGS BR rats at dose levels of 0 (control), 30, 100, or 300 mg/kg/day. Female rats were dosed for four weeks premating, through mating, gestation, and lactation (a total of 55 to 63 days of treatment). Male rats were treated for 55 days (four weeks premating and through mating). Premating, body weights, food consumption, and clinical signs were recorded. Hematology, clinical chemistry, and urine analyses were conducted at the end of the premating period. A neurobehavioral test battery was conducted prior to and after four weeks of treatment. After the premating period, females were paired with males from the same groups for 1-2 weeks. Litters were delivered, pups were evaluated for structural integrity, and pup body weights were recorded on days 0 and 4 postpartum. Lactating females and their offspring were sacrificed on postpartum day 4. Selected organs were weighed and the tissues were examined microscopically from the lactating females. Offspring were examined for clinical abnormalities. A test substance-related reduction in body weight gain occurred in male rats administered 300 mg/kg/day. Decreased body weight gain in the 300 mg/kg/day males was accompanied by increased food consumption and decreased food efficiency. Females administered 100 or 300 mg/kg/day had test substance-related, significantly decreased body weight and body weight gain during gestation, that was accompanied by a significant increase in food consumption (300 mg/kg/day group only), and significantly decreased food efficiency. There were no test-substance related effects on clinical observations in males or females during the premating phase, or in females during gestation or lactation. Neurobehavioral parameters and motor activity were unaffected by CDDT-treatment. During this study, statistically significant treatment-related changes were observed in several clinical pathology parameters. The decreases in red cell mass (RBC, HGB, HCT) were minimal and, due to the magnitude, were not expected to result in biological effects. Similarly, minimally increased potassium and mildly decreased triglycerides were not of a magnitude to be biologically significant. Finally, changes in serum enzymes (AST, ALT, ALP), urea nitrogen, and serum protein occurred in directions that are not associated with toxicity. The changes in urine volume, urine concentration, and urea nitrogen may be the result of elevated glomerular filtration rate and altered tubular fluid flow, in the absence of any histopathological change. No effects on reproduction in parental males or females were produced by CDDT. Body weights of pups in the 300 mg/kg group were significantly decreased on postpartum days 0 and 4. There were no test-substance related effects on clinical observations, number of pups born, and the number of pups born alive, or the number of pups surviving through lactation day 4. The no-observed-adverse-effect level (NOAEL) for CDDT was 30 mg/kg/day based on decreased body weight and body weight gain, increased food consumption, and decreased food efficiency in females administered 100 or 300 mg/kg/day. The NOEL in pups was 100 mg/kg/day, based on decreased body weights of pups in the 300 mg/kg/day group during lactation.

Inhalation toxicity of cyclododecatriene in rats.

Cyclododecatriene (CDDT, CAS No. 4904-61-4) was tested for its inhalation toxicity in rats following repeated exposures. Male rats were exposed nose-only to CDDT for 6 hr/day, 5 days/wk for a total of 9 exposures over 2 weeks. Particular attention was paid to neurotoxicologic endpoints. Concentrations of 0 (control), 5, 50, and 260 ppm were studied. The 260 ppm chamber contained both vapor and aerosol while the 5 and 50 ppm chambers were vapor only. Four groups of 10 rats each were used to measure standard clinical signs and growth, clinical pathology (including hematology, biochemistries, and urine analysis), and tissue pathology. Another 4 groups of similar size were used for neurotoxicity testing. In the standard toxicity groups, 1/2 of the rats were sacrificed 1 day following the 9th exposure; the other half underwent a 2-week recovery period prior to being sacrificed (recovery group). During the exposures rats inhaling 260 ppm had a diminished or absent response to an alerting stimulus. Irregular respiration and lethargy were observed in these rats immediately following exposure. These signs were rapidly reversible and were not seen prior to the subsequent exposure. Body weights in rats exposed to either 50 or 260 ppm were significantly lower than the corresponding controls. No compound-related clinical pathology changes were seen in any of the test groups and tissue pathology effects only occurred in the nasal tissue. In rats exposed to 260 ppm, minimal degeneration/necrosis of nasal olfactory epithelium was observed in rats examined immediately following the exposure period. This change was not seen in the recovery rats. Functional observational battery (FOB) assessments and motor activity (MA) evaluations conducted after the 4th and 9th exposures on rats from all test groups, and specific neuropathologic evaluation on perfused brain, spinal cord, and skeletal muscle from rats exposed to 260 ppm failed to demonstrate any specific neurotoxicity. Outward signs of sedation were seen at the top level tested. Under the conditions of this test, the no-observed-adverse-effect level (NOAEL) was determined to be 5 ppm based upon a reduced rate of body weight gain in the 50 ppm group. No specific neurotoxicity was detected and the histopathologic response was limited to reversible changes in the nasal epithelia in rats exposed to 260 ppm.