Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green.

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.

Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green.

Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green. To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks. The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks. Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green. A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed. We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green. Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat. These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

Food borne microbial pathogens of cultured aquatic species.

This article provides a broad overview of microbial pathogens associated with marine and fresh water aquatic animals, including Vibrio species, Escherichia coli, Streptococcus iniae, Salmonella species, and Edwardsiella tarda. Historically, cultured fish were not considered important vectors of human pathogens. This situation is changing, partly due to increasing animal densities as a consequence of a rapidly growing industry and partly due to increasing awareness by health care providers of pathogens in aquatic species that may result in human illness. Concerns facing the industry are also discussed along with possible solutions.

Public health issues in aquaculture.

The authors address the public health issues associated with the consumption of aquacultural products using numerous examples from the United States of America. As with other foods, public health risks exist but these mostly involve open water environments or products which are consumed raw or undercooked. Unlike wild fisheries, inland aquaculture systems can minimise public health risks by proper site evaluation and good aquacultural practices. Responsible use of pesticides and therapeutants can prevent violative residues to assure product safety and wholesomeness. The implementation of hazard analysis and critical control point regulations will further enhance the preventive approach to hazards control. The most challenging public health risks arise from shellfish production in open, surface waters, where both naturally-occurring and trace environmental residue contaminants can bioaccumulate in tissues and may cause disease outbreaks (and, in severe cases, death). Water quality certification programmes and field surveillance efforts including product sampling, testing and monitoring can address critical safety criteria. This paper focuses primarily on public health risks associated with production: however, the fact that consumer risks also occur as a result of the processing of aquacultural products and that foodborne diseases arise additionally from unsanitary handling or preparation and storage at incorrect temperatures (as is the case for food products from other animals) must also be taken into consideration.

Pharmacokinetics, bioavailability, plasma protein binding and disposition of nalidixic acid in rainbow trout (Oncorhynchus mykiss).

1. The pharmacokinetics, disposition and bioavailability of nalidixic acid were examined in Rainbow Trout following i.v. and per os administration (5 mg/kg). 2. Nalidixic acid was biexponentially eliminated from plasma following i.v. dosing (t1/2 alpha = 0.06 h, t1/2 beta = 23.0 h). The volume of distribution (Vss) and total body clearance (Clb) were 964.7 ml/kg and 31.5 ml/kg/h, respectively. 3. In vitro plasma protein binding was specific and saturable over a range of concentrations from 0.43 microM to 20.0 mM. Binding was approx. 26% at kinetically relevant plasma concentrations. 4. Apparent oral bioavailability was determined to be > 100%, suggesting that nalidixic acid was largely bioavailable and non-linear pharmacokinetics were evoked. 5. Oral studies demonstrated the highest 14C nalidixic acid equivalent concentrations in bile, intestine and liver. Muscle contained intermediate concentrations but among all organs accounted for the greatest total amount of drug (12.2% of dose). Mass balance studies demonstrated composite values for per cent of dose administered of 23.7, 18.8, 8.5, 10.0, 7.4 and 2.3% for 1, 2, 3, 6, 9 and 15 days, respectively. 6. A glucuronic acid conjugate of nalidixic acid was identified by n.m.r. and mass spectral analysis as the single primary metabolite.

Effect of halothane, isoflurane, and pentobarbital anesthesia on myocardial irritability in chickens.

The relative myocardial irritant properties of halothane, isoflurane, and pentobarbital were evaluated in chickens. Sixteen adult male broiler chickens were randomly assigned to 1 of 3 groups: group-1 chickens were anesthetized with pentobarbital (30 mg/kg, IV), group-2 chickens were anesthetized with halothane (end tidal halothane 1.2%), and group-3 chickens were anesthetized with isoflurane (end tidal isoflurane 2.1%). Birds in any 2 of the 3 treatment groups were tested on any 1 day. Local anesthesia was induced, and blood pressure, heart rate, ECG, and blood gas variables were measured before general anesthesia was induced. Positive-pressure ventilation with an inspired O2 fraction greater than 0.95 was adjusted to result in an end tidal CO2 concentration that reflected a Paco2 similar to that obtained prior to anesthesia and ventilation. All measurements were repeated. The threshold for ventricular fibrillation in response to electrical stimulation of the heart was then determined for all birds. Effects of anesthesia on hemodynamic and blood gas variables were similar in all 3 groups. Compared with halothane or pentobarbital, isoflurane anesthesia resulted in a significantly (P less than 0.05) lower threshold for electrical fibrillation of the heart.

Effect of age and growth rate on myocardial irritability in broiler chickens.

Young broiler chickens are undergoing a period of extremely rapid growth and may be expected to be in a state of extreme endocrine and biochemical flux. These birds are also subject to a sudden death syndrome of unknown etiology. We hypothesized that an increased myocardial sensitivity in birds exhibiting early rapid growth may contribute to this syndrome. The objective of the current study was to investigate the interaction between early growth rate and age on myocardial irritability in young broiler chickens. This study utilized 74 male broiler chickens between 3 (Group A, 21-24 days) and 6 (Group B, 43-47 days) weeks of age exhibiting rapid (heavy weight, greater than 425 g at 2 weeks) and slow (low weight, less than 350 g at 2 weeks) early growth. Physiologic parameters such as blood pressure, heart rate, and arterial blood gases in the awake, restrained birds were essentially unchanged across these groups. Myocardial irritability of the anesthetized bird (pentobarbital) as measured by the threshold to electrical fibrillation was significantly increased only in the Group A HW birds. The results of this study suggest an increased myocardial irritability in large young broilers (3 weeks of age) that is no longer present in a similar group of older birds (6 weeks of age). These findings are consistent with the hypothesis that there is an increased myocardial sensitivity in birds succumbing to sudden death syndrome, with death due to myocardial fibrillation. Further research is needed to determine the mechanisms involve.

Induced pasteurellosis and pulmonary vascular adrenoceptor function in bull calves.

Effects of Pasteurella haemolytica inoculation on pulmonary vascular function were studied in 5 conscious standing Jersey bull calves. Instruments were implanted in each calf to measure pulmonary arterial, pulmonary arterial wedge, left atrial, and systemic arterial pressures and cardiac output. Each calf was challenge exposed with 5 sequential 3-minute infusions of isoproterenol (a beta agonist) or phenylephrine (an alpha agonist) for maximal doses of 1.8 micrograms of isoproterenol or 2.3 micrograms of phenylephrine/kg of body weight/min. The calf was allowed 1 hour to recover, was anesthesized, and administered a 20-ml intratracheal infusion of live P haemolytica (10(6) colony-forming units/ml) followed by a 20-ml saline flush. The pulmonary hemodynamic response to isoproterenol and phenylephrine was examined again in each calf 4 days later. Calves developed a pneumonic pasteurellosis involving 26 to 43% of the lungs. There was a significantly (P less than 0.05) reduced resistance in the pulmonary arterial compartment after inoculation. Isoproterenol infusion decreased resistance in the pulmonary arterial, pulmonary venous, and systemic vascular compartments. The decrease in the pulmonary venous compartment in response to isoproterenol was significantly (P = 0.01) smaller after P haemolytica inoculation. After administration of 1.8 micrograms of isoproterenol/kg/min, resistance in the pulmonary venous compartment was 0.90 +/- 0.22 (mean +/- SD) before and 1.25 +/- 0.39 after Pasteurella inoculation. Phenylephrine resulted in an increase in pulmonary arterial, pulmonary venous, and systemic vascular compartments. There was a mild (P = 0.08) decrease in the pulmonary arterial compartment response to phenylephrine. Seemingly, Pasteurella inoculation blunted beta-receptor function in the pulmonary vascular bed, mainly in the veins, contributing to edema.

Adrenoceptor function in the bovine pulmonary circulation.

The bovine pulmonary vascular response to alpha- and beta-agonists was studied using an awake intact calf model. Pulmonary arterial pressure, pulmonary arterial wedge pressure, left atrial pressure, systemic arterial pressure, and cardiac output were measured in response to 3 min infusions of isoproterenol (beta-agonist; 0.12, 0.24, 0.48, 0.9, and 1.8 micrograms X kg-1 X min-1) and phenylephrine (alpha-agonist, 0.15, 0.30, 0.60, 1.15, and 2.30 micrograms X kg-1 X min-1). Phenylephrine caused an increase in vascular resistance in the pulmonary arterial and venous compartments. The slope of the resistance in response to phenylephrine was greater in the pulmonary arterial than pulmonary venous circulation. Isoproterenol resulted in a dose-dependent decrease in vascular resistance in the pulmonary arteries and veins. The vascular resistance was decreased to the same level in the pulmonary arteries and veins although the arteries showed a greater percent change. In addition, isoproterenol infusion resulted in a transient decrease in arterial pH and increase in values for packed cell volume and haemoglobin.

Pulmonary vascular responsiveness in cold-exposed calves.

The pulmonary vascular responses to acute hypoxia and to infusions of histamine and 5-hydroxytryptamine (5-HT) were recorded in unanesthetized standing bull calves under neutral (16-18 degrees C) and cold (3-5 degrees C) temperature conditions. Cold exposure alone resulted in a significant increase in pulmonary arterial wedge pressure from 10.2 +/- 3.5 to 15.9 +/- 4.9 Torr (1 Torr = 133.322 Pa). Resistance to blood flow between the pulmonary wedge and the left atrium significantly increased from 0.50 +/- 0.51 to 1.21 +/- 0.78 mmHg . L-1 . min-1 (1 mmHg = 133.322 Pa) with cold exposure. This apparent pulmonary venoconstrictor response to cold exposure was further evaluated to determine if hypoxia, histamine, or 5-HT responsiveness was altered by cold exposure. Twelve minutes of hypoxia increased pulmonary arterial and systemic arterial pressures, heart rate, and respiratory rate similarly in cold and neutral temperatures. Cold exposure did not alter the dose-related reductions of systemic arterial and pulmonary arterial pressures in response to histamine. Similarly, the decreases in systemic arterial pressure and heart rate and increases in pulmonary arterial and left atrial pressures in response to 5-HT were not significantly different in cold and neutral conditions. It was concluded that acute, mild cold exposure results in an increase in resistance to blood flow in the pulmonary venous circulation without a general increase in pulmonary vascular reactivity, as measured by responses to hypoxia, histamine, and 5-HT.

Pulmonary vascular changes in young and aging rats exposed to 5,486 m altitude.

Young (YNG) and middle-aged (MA) male rats were exposed to 5,486 m for durations ranging from 1 to 42 days to determine the effect of age on the progression of polycythemia, right ventricular hypertrophy (RVH), lung vascular muscularization, and pulmonary vascular responsiveness. Other rats were exposed for 42 days at 5,486 m and were then allowed to recover at 1,520 m for periods up to 42 days. The progression and subsequent regression of polycythemia and RVH with altitude exposure were similar for YNG and MA rats. However, YNG rats exhibited vascular muscularization during the altitude exposure, characterized by hypertrophy of smooth muscle cells, whereas MA rats exhibited little or no change in vascular morphology. Lungs from both altitude-exposed YNG and MA rats exhibited blunting of acute hypoxic pulmonary vasoconstriction upon exposure to 5,486 m, with more severe blunting apparent in MA rats. Pressor responses to angiotensin II (AII) were potentiated in lungs from high altitude rats, particularly in the YNG rats, and this increased responsiveness persisted during the recovery period. A positive correlation was found in YNG rats between the degree of vascular muscularization and the pressor response to AII, suggesting that increased muscle mass was partially responsible for the potentiated AII responses. However, MA rats did not exhibit the same correlation for AII, and neither YNG nor MA rats exhibited increased responsiveness to 5-hydroxytryptamine. The results indicate that age influences the morphologic responses to altitude exposure and vascular responsiveness to AII, but does not affect the polycythemic response or the degree of RVH.

Pulmonary and systemic vascular responses to tolazoline in neonatal and mature calves.

Tolazoline was infused intravenously (2 mg/kg over 2 minutes) in awake neonatal and mature calves. In normoxic pulmonary normotensive neonatal calves, tolazoline induced minimal hemodynamic changes, except for an immediate bradycardia. Tolazoline caused increases in systemic arterial pressure and vascular resistance and a bradycardia in normoxic normotensive mature calves. Calves were also made hypoxic, by breathing 12% O2 by facemask, in order to produce pulmonary hypertension. In hypoxic pulmonary hypertensive neonatal calves, tolazoline induced transient pulmonary vasodilation, a reduction in systemic arterial pressure, and marked bradycardia. Similarly, tolazoline caused reductions in pulmonary and systemic arterial pressure and bradycardia in hypoxic pulmonary hypertensive mature calves. Pentobarbital anesthesia in neonatal calves induced marked hemodynamic changes but did not alter the cardiovascular responses to tolazoline. A significant correlation was found between the baseline pulmonary arterial pressure and the magnitude of the pulmonary depressor response to tolazoline when all interventions were evaluated. Thus, tolazoline induced transient pulmonary vasodilation in pulmonary hypertensive calves but simultaneously caused profound transient bradycardia, particularly in neonatal calves.

Hypoxic pressor responses in lungs from rats acutely exposed to simulated high altitude.

Male Sprague-Dawley rats were exposed to 6, 3 or 1 h of simulated high altitude (380 Torr). The isolated perfused lung of each animal was challenged with 4 sequential periods of hypoxia, angiotensin II (AII), and 5-hydroxytryptamine (5-HT) following altitude exposure. Isolated perfused lungs of low altitude control rats were similarly challenged. The acute hypoxic vasoconstrictor response was significantly reduced in animals exposed to 3 and 6 h of high altitude compared to low altitude control animals. The hypoxic pressor response was not significantly different from control in animals exposed to 1 h of simulated altitude. Pressor responses to AII and 5-HT following altitude exposure were not significantly different from controls.

Altered vascular responsiveness in isolated perfused lungs from aging rats.

Systemic vascular hyporesponsiveness has been well documented in aged rats, but a similar decreased vasoreactivity of the pulmonary vasculature has not been reported. Isolated lungs from young (3-5 months) and middle-aged (12-14 months) rats were perfused with whole blood and challenged alternately with alveolar hypoxia, angiotensin II, and 5-hydroxytryptamine. Hypoxic pulmonary vasoconstriction was less in the aging rats during both 5-min and 10-min hypoxic exposures. Likewise, log-dose response curves for angiotensin II and 5-hydroxytryptamine were right-shifted in the aging rats, indicating decreased responsiveness to the vasoactive agents. Since the vascular responses to all three vasoconstrictors were lower in the older animals, a generalized pulmonary vascular hyporeactivity with advancing age is suggested.

Pulmonary and systemic vascular actions of tolazoline in anesthetized dogs.

The cardiovascular actions of tolazoline are poorly understood. Therefore, we administered tolazoline (2 mg kg-1, IV) to anesthetized adult dogs and puppies. Tolazoline induced transient pulmonary and systemic pressor responses, transient systemic vasoconstriction and pulmonary vasodilation, and transient hypoxemia and acidosis in pentobarbital anesthetized dogs. None of these responses were evident during a second injection of tolazoline, indicating either the development of tachyphylaxis or a secondary antagonistic property of tolazoline. These responses to tolazoline were also prevented by alpha adrenergic blockade. Histamine H1 - and H2-receptor blockade sustained the transient pulmonary and systemic pressor and systemic vasoconstrictor responses to tolazoline. Thus, tolazoline appears to activate both alpha and histamine receptors in the pulmonary and systemic vascular beds of the anesthetized dog. In anesthetized puppies, tolazoline induced a slight pulmonary pressor response, marked bradycardia, and systemic hypertension. The present findings confirm the pharmacological complexity of tolazoline and suggest that anesthesia, age, and pulmonary vascular tone are important factors in determining the cardiovascular responses to tolazoline.