Lynn P. Weber

Increased kidney, liver, and testicular cell death after chronic exposure to 17α-ethinylestradiol in medaka (Oryzias latipes)

Sublethal effects observed in fish exposed to environmental estrogens may be mediated via stimulation of cell death. To investigate whether cell death is induced in fish after chronic exposure to estrogenic chemicals, Japanese medaka (Oryzias latipes) were exposed from hatch until sexual maturity to 10 ng/L 17alpha-ethinylestradiol (EE2) or acetone solvent (control). Cell death was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl-mediated dUTP nick end-labeling (TUNEL), which labels nuclei of cells containing apoptotic or necrotic (fragmented) DNA. The major impact of EE2 exposure in both male and female medaka was to significantly increase the number of TUNEL-positive hepatocytes and kidney tubule cells compared to control. Cell morphology was consistent with apoptosis in the liver and cloudy swelling or necrosis in the tubule cells. The number of TUNEL-positive interstitial (hematopoietic) and glomerular cells was significantly greater in the kidneys of EE2-exposed male, but not female, medaka. The EE2 exposure also significantly increased the number of TUNEL-positive testicular cells in medaka compared to corresponding controls, namely Leydig cells, Sertoli cells, spermatocytes, and spermatids. In medaka with gonadal intersex, areas of fibrosis and areas containing female gonadal cells were relatively unstained with TUNEL. No effect of EE2 exposure on the number of TUNEL-positive ovarian somatic cells or on the rate of female ovarian follicle atresia was found. These results suggest that chronic exposure to EE2 in medaka is hepatotoxic and nephrotoxic in both sexes, whereas gonadal toxicity is specific to males.

Hepatic and vascular mRNA expression in adult zebrafish (Danio rerio) following exposure to benzo-a-pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system are not clear. We hypothesized that AhR-mediated changes in adult vascular tissue gene expression would differ from that of hepatic tissue. Therefore, zebrafish (Danio rerio) were intraperitoneally injected with the AhR agonists benzo-a-pyrene (BaP; 1mg/kg) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 20microg/kg) alone and in combination with the AhR antagonists resveratrol (Res; 10mg/kg) or alpha-naphthoflavone (ANF; 50mg/kg). Hepatic and mesenteric artery cytochrome P450 enzyme (subtypes 1A, 1B1, 1C1, and 1C2) and cyclooxygenase enzyme (subtypes 1, 2a, and 2b) mRNA expression was quantified using real-time reverse transcriptase PCR. TCDD exposure significantly increased (p<or=0.05 in Tukeys posteriori test after 1-way ANOVA; n=4-6/group) CYP1A, CYP1C1, and COX-2b mRNA expression in hepatic tissue (105+/-21, 12+/-2, and 2+/-0.3 fold-increase, mean+/-SEM respectively). TCDD also increased CYP1A, CYP1B1, CYP1C1, CYP1C2, and COX-1 mRNA expression in mesenteric artery (121+/-23, 5+/-1, 28+/-6, 7+/-1, and 3+/-0.3, respectively). Importantly, while BaP exposure elicited no significant alterations in hepatic CYP mRNA expression, it increased COX-1 and COX-2b in liver tissues (3+/-1 and 2+/-0.1, respectively), as well as CYP1A, CYP1B1, CYP1C1, CYP1C2, and COX-1 expression in mesenteric artery (2+/-0.3, 4+/-0.3, 5+/-1, 5+/-1, and 2+/-0.3, respectively). Resveratrol was able to antagonize TCDD-induced CYP1C2 in mesenteric artery but was without effect in all other treatments in both liver and mesenteric artery. In contrast, ANF antagonized TCDD and BaP-induced COX-2b and TCDD-induced CYP1C1 expression increases, as well as reduced baseline CYP1B1 and COX-2a expression in liver, while failing to affect BaP and TCDD-induced hepatic CYP1A increases. However, in mesenteric artery, ANF alone acted instead as an agonist to increase expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, COX-2a and COX-2b. Thus, there are important differences in response to both AhR agonists and antagonists between liver and mesenteric artery in adult zebrafish. The vascular-specific changes in gene expression will be linked to future studies examining alterations in cardiovascular function produced by acute AhR agonist exposure in adult fish.

Postprandial impairment of flow-mediated dilation and elevated methylglyoxal after simple but not complex carbohydrate consumption in dogs

Hyperglycemia produces oxidative stress, which may impair endothelial function. Methylglyoxal, a reactive intermediate metabolite of glucose, is known to cause oxidative stress and is produced when excess carbohydrate is consumed in diabetic patients, but postprandial responses in healthy patients are unknown. We hypothesize that methylglyoxal levels will cause impaired endothelial function via increased oxidative stress after consuming a high glycemic index meal in healthy animals. Normal-weight laboratory beagles (n = 6) were used in a crossover study that tested postprandial responses of 4 complex carbohydrate sources (barley, corn, peas, rice) vs a simple carbohydrate (glucose). Blood samples were taken prefeeding and at timed intervals after feeding to measure serum glucose, insulin, nitrotyrosine, and methylglyoxal. Flow-mediated dilation (FMD), cardiac function (echocardiography), and blood pressure measurements were determined before and 60 minutes after feeding. The mean (±SEM) glycemic indices of the complex carbohydrate sources were 29 ± 5 for peas, 47 ± 10 for corn, 51 ± 7 for barley, and 55 ± 6 for rice. Postprandial FMD was lowest in the glucose group and significantly different from both the corn group and the FMD value for all complex carbohydrates combined. Methylglyoxal was significantly elevated at 60 minutes postprandial after glucose compared with the other carbohydrate sources. No significant effects of carbohydrate source were observed for blood pressure, nitrotyrosine, or echocardiographic variables. The novel finding of this study was that methylglyoxal levels increased after a single feeding of simple carbohydrate and may be linked to the observed postprandial decrease in endothelial function. Thus, consuming low-glycemic-index foods may protect the cardiovascular system by reducing oxidative stress.

Persistent effects on adult swim performance and energetics in zebrafish developmentally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) remains a potent and persistent toxicant in aquatic environments, causing lethal developmental deformities in fish. However, few studies have examined sublethal or persistent effects of developmental TCDD exposure and none have examined its effects on swimming capabilities in sub-adult fish. The objective of the current study was to examine whether effects of TCDD exposure during the critical period of cardiovascular development (2-4 days post fertilization) on swim performance, triglyceride stores and cardiovascular deformities would persist until adulthood in zebrafish. Zebrafish larvae were exposed between 48 and 96 h post fertilization to 1, 0.1, 0.01 ng/L TCDD or DMSO control (0.005%), then raised in clean water for 90 days. Despite having equal survivability, no significant increase in gross deformities and no change in cytochrome P450 1A (CYP1A) activity was observed, while critical swimming speed and dorsal aorta diameter were significantly decreased in TCDD-exposed fish at 90 days. Furthermore, whole body triglycerides were significantly elevated in TCDD-exposed fish both before and after swim testing. Therefore sublethal TCDD exposure during zebrafish development caused a persistent decrease in swim endurance. The cause of this persistent decrease in swim endurance is not known, but may be related to behavioral adaptations limiting swimming capabilities, failure to mobilize triglyceride stores, vascular deformities limiting blood flow to the periphery, or a combination of these factors.

Intranasal benzo[a]pyrene alters circadian blood pressure patterns and causes lung inflammation in rats.

Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are environmental contaminants formed during organic material combustion (e.g. burning fossil fuels and cigarette smoke). BaP toxicity is mediated, in part, by activation of the aryl hydrocarbon receptor and formation of reactive metabolites, both of which lead to increased oxidative stress. Since air pollution and cigarette smoking are known to increase cardiovascular disease in humans, the objective of this study was to determine the effects of 7-day intranasal BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Arterial pulse wave dP/dt was used an indicator of arterial stiffness and was compared to both functional (nitric oxide production and bioactivity, endothelin-1 levels) and structural (wall thickness) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic cytochrome P450 1A1 (CYP1A1) activity. BaP exposure altered the circadian pattern of blood pressure, with a reduction in the normal dipping pattern during sleep. This was associated with increased neutrophil recruitment in the lungs of BaP-exposed rats. In contrast, BaP had no effect on cardiovascular tissue histology, arterial stiffness, oxidative stress or lung and liver CYP1A1 activity. Thus, the current study does not support the hypothesis that BaP reactive metabolites increase oxidative stress leading to reduced vascular NO bioactivity and increased blood pressure. Instead, the current study suggests that inflammation, detected only in the lung, is associated with altered circadian rhythm of blood pressure.

Overwinter alterations in energy stores and growth in juvenile fishes inhabiting areas receiving metal mining and municipal wastewater effluents

The winter stress syndrome hypothesis proposes that the combination of winter conditions and contaminant exposure reduces overwinter survival in juvenile fishes, mainly due to increased depletion of stored energy (lipids). To test this hypothesis in the field, juvenile fathead minnows (Pimephales promelas), creek chub (Semotilus atromaculatus), and white sucker (Catostomus commersoni) were collected from three exposure sites along Junction Creek, Sudbury, Ontario, Canada, representing cumulative inputs from metal mining and municipal wastewater. Overwinter survival potential was determined through measurements of growth (length, weight, muscle RNA/DNA ratio, muscle proteins) and energy stores (whole body triglycerides) in fish collected just prior to and following the overwinter period. We hypothesized that fish collected from exposure sites would exhibit reduced growth and energy storage compared to reference fish in both fall and spring, and that fish from all sites would exhibit reduced energy storage in spring compared to the previous fall. Whole body Se concentrations were elevated (11-42 microg/g dry wt) in juvenile fathead minnows and white sucker collected at two exposure sites in comparison to fish collected from the reference site (3-6 microg/g dry wt). In contrast to our hypothesis, fathead minnows were larger with greater triglyceride stores at exposure sites compared to the reference site. White suckers were smaller at exposure sites but did not differ in triglycerides among sites. Overall, the results in these fish species exposed to metal mining and municipal wastewaters do not support the winter stress syndrome hypothesis. It is recommended that future studies focus on relating growth and energy storage with other environmental factors such as habitat and food availability in addition to anthropogenic contamination.

Acute exposure to 2,4-dinitrophenol alters zebrafish swimming performance and whole body triglyceride levels

While swimming endurance (critical swimming speed or U(crit)) and lipid stores have both been reported to acutely decrease after exposure to a variety of toxicants, the relationship between these endpoints has not been clearly established. In order to examine these relationships, adult zebrafish (Danio rerio) were aqueously exposed to solvent control (ethanol) or two nominal concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24-h period. Following exposure, fish were placed in a swim tunnel in clean water for swimming testing or euthanized immediately without testing, followed by analysis of whole body triglyceride levels. U(crit) decreased in both the 6 mg/L and 12 mg/L DNP groups, with 12 mg/L approaching the LC₅₀. A decrease in tail beat frequency was observed without a significant change in tail beat amplitude. In contrast, triglyceride levels were elevated in a concentration-dependent manner in the DNP exposure groups, but only in fish subjected to swimming tests. This increase in triglyceride stores may be due to a direct interference of DNP on lipid catabolism as well as increased triglyceride production when zebrafish were subjected to the co-stressors of swimming and toxicant exposure. Future studies should be directed at determining how acute DNP exposure combines with swimming to cause alterations in triglyceride accumulation.

Comparison of the acute effects of benzo-a-pyrene on adult zebrafish (Danio rerio) cardiorespiratory function following intraperitoneal injection versus aqueous exposure

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. PAH exposure causes developmental toxicity in multiple fish species, while acute adult fish toxicity is thought to be minimal. The literature increasingly suggests sublethal PAH effects may occur, but differences in exposure route may confound conclusions. We hypothesized that acute PAH exposure in adult fish will cause cardiorespiratory impairment that will not differ with exposure route. In order to investigate this hypothesis, adult zebrafish (Danio rerio) were injected intraperitoneal (i.p.) twice with increasing concentrations of the prototypical PAH, benzo-a-pyrene (BaP; 0.1, 10, and 1000μg/kg) or exposed aqueously (static, renewal at 24h; 16.2 and 162μg/L) for 48h and compared to corresponding dimethylsulfoxide controls. No mortalities or significant effects on weight of the fish were noted at any exposure concentration or route. At 48h, fish were subjected to swimming tests with concurrent oxygen consumption measurement (n=10 fish/treatment) or echocardiography (n=12 fish/treatment). Oxygen consumption (MO2) was increased at three swimming speeds in BaP-injected groups compared to control (p<0.01 in Fishers LSD tests after two-way ANOVA). In contrast, aqueously BaP-exposed fish showed increased MO2 under only basal conditions. Despite increased oxygen demand, ventricular heart rate was significantly decreased in BaP-exposed fish, both injected and aqueously-exposed. Analysis of BaP body burdens in fish tissue allowed for identification of an overlapping dose group between exposure routes, through which comparisons of cardiorespiratory toxicity were then made. This comparison revealed most effects were similar between the two exposures routes, although minor differences were noted. At similar BaP body burdens, injected fish suffered from more severe bradycardia than aqueously exposed fish and had greater levels of increases in cytochrome P4501A (CYP1A) mRNA levels in liver and heart tissue compared to aqueous exposed fish. In conclusion, acute BaP exposure in adult zebrafish had negative effects on cardiorespiratory function. Differences in effect between exposure routes were attributed primarily to differences in bioavailability, since overall, similar effects were noted between the two exposure routes when similar BaP body burdens were achieved.

Secondhand tobacco smoke, arterial stiffness, and altered circadian blood pressure patterns are associated with lung inflammation and oxidative stress in rats

Chronic smoking and secondhand tobacco smoke exposure are major risk factors for cardiovascular disease that are known to adversely alter the structural and mechanical properties of arteries. The objective of this study was to determine the effects of subchronic secondhand tobacco smoke exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in conscious, unsedated radiotelemetry-implanted rats. Pulse wave change in pressure over time (dP/dt) was used an indicator of arterial stiffness and was compared with both structural (wall thickness) and functional (nitric oxide production and bioactivity and endothelin-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver was examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450, specifically CYP1A1). Subchronic secondhand tobacco smoke exposure altered the circadian pattern of heart rate and blood pressure, with a loss in the normal dipping pattern of blood pressure during sleep. Secondhand tobacco smoke exposure also increased pulse wave dP/dt in the absence of any structural modifications in the arterial wall. Furthermore, although nitric oxide production and endothelin-1 levels were not altered by secondhand tobacco smoke, there was increased inactivation of nitric oxide as indicated by peroxynitrite production. Increased lung neutrophils or pulmonary CYP1A1 may be responsible for the increase in oxidative stress in rats exposed to secondhand tobacco smoke. In turn, this may be related to the observed failure of blood pressure to dip during periods of sleep and a possible increase in arterial stiffness.

Resveratrol preserves cardiac function, but does not prevent endothelial dysfunction or pulmonary inflammation after environmental tobacco smoke exposure

The mechanisms by which environmental tobacco smoke (ETS) causes adverse cardiovascular effects remain unclear. Resveratrol is a natural polyphenol from red wine which may be beneficial to the cardiovascular system. Therefore, the ability of daily oral resveratrol (5mg/kg) to prevent adverse effects of a 14-day ETS exposure (1 h/day) on endothelial function (flow-mediated dilation), left ventricular function (echocardiography) and blood pressure (oscillometry) was assessed in juvenile male pigs (n=4 pigs/group). After a 14-day exposure to ETS, flow-mediated dilation was impaired while plasma nitrotyrosine was increased compared to sham-exposed pigs indicating impaired endothelial function. In ETS-exposed pigs, plasma C-reactive protein levels, lung cytochrome P4501A1 activity, bronchoalveolar lavage fluid total white blood cell count and leukocyte elastase activity were all significantly increased compared to sham-exposed pigs. Resveratrol treatment failed to prevent most ETS-mediated effects examined, but did increase left ventricular end-diastolic volume and ejection fraction in the presence of ETS exposure. In summary, ETS exposure impaired endothelial function and increased oxidative stress which was associated with pulmonary and systemic inflammation, but resveratrol failed to protect against these changes. More importantly, resveratrol exerted a positive effect on left ventricular function which may help explain the French paradox.