Changes in social and feeding behaviors, activity, and salivary serum amyloid A in cows with subclinical mastitis.

The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors, such as decreased activity, feeding and drinking (here labeled "core maintenance" behaviors), and less well-studied social, grooming, and exploratory behaviors (here labeled "luxury" behaviors). Luxury behaviors are biologically predicted to change at lower levels of mastitis infection and are, therefore, particularly relevant to detecting subclinical mastitis. Salivary serum amyloid A is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for 17 matched pairs of commercial barn-housed Holstein-Friesian cows. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC; SCM: SCC >200 × 1,000 cells/mL; CTRL: SCC <100 × 1,000 cells/mL). SCM cows were selected for study ad hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 d: SCC (d 1), behavior (d 2), salivary SAA (d 3). All behaviors performed by the focal cows over a single 24-h period were coded retrospectively from video footage, and differences between the SCM and CTRL groups were investigated using the main data set and a subset of data corresponding to the hour immediately following morning food delivery. Saliva was collected using cotton swabs and analyzed for SAA using commercially available ELISA kits. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows displayed reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic noncontact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. The SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with 2 conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings.

An immunological assessment of group-housed rabbits.

Laboratory rabbits kept in barren 'traditional' cages tend to develop stereotypic behaviours and bone deformities. We have used an alternative regime, housing adult does as groups of four or five in floor pens (2.5-3 m2) supplied with hiding places and bedding. High- and low-ranking members of each group were identified, and their immunological status compared in terms of blood leucocyte function (chemiluminescence and mitogen tests), complement activity, and antibody production to soluble and cellular antigens. We found no evidence of immunosuppression, either in groups of a 'docile' breed (New Zealand White) or Dutch crosses. These results, together with the animals' general health and ease of handling, lead us to conclude that group-housed does are suitable for raising antisera and other purposes, provided that they are adequately monitored.

Refinement and verification of the physiologically based dosimetry description for acrylonitrile in rats.

The physiologically based dosimetry description for acrylonitrile (ACN) and its mutagenic epoxide metabolite 2-cyanoethylene oxide (CEO) in F-344 rats (M. L. Gargas, M. E. Anderson, S.K.O. Teo, R. Batra, T. R. Fennell, and G. L. Kedderis, 1995, Toxicol. Appl. Pharmacol. 134, 185-194) has been refined to include a physiological stomach compartment and the reactions of ACN with tissue glutathione (GSH). The second-order rate constant for reaction of ACN and GSH at pH 7.3 was measured and included in the dosimetry description. Metabolic parameters for ACN and CEO were estimated from oral bolus pharmacokinetic studies and previously obtained iv bolus data (3.4, 47, 55, or 84 mg ACN/kg). Rats were given bolus oral doses of 3, 10, or 30 mg ACN/kg in water, and blood samples were collected at selected time points. ACN and CEO blood concentrations were determined by gas chromatography. The brain and liver concentrations of ACN and CEO were also measured after 10 mg ACN/kg po. ACN elimination from blood was described by saturable P450 epoxidation (Vmax of 5.0 mg/hr/kg and K(M) of 1.5 mg/liter) and first-order GSH conjugation (73 hr(-1)/kg). CEO elimination was described by first-order GSH conjugation (500 hr(-1)/kg). The pharmacokinetic data were well simulated, although CEO blood concentrations after bolus oral dosing were somewhat overestimated. Sensitivity analysis of the dosimetry description indicated that the inhalation exposure route was much more sensitive to changes in metabolic and physiological parameters than either the iv or oral bolus routes. Therefore, inhalation pharmacokinetic data were obtained and compared to simulations of the dosimetry description. Rats were exposed to 186, 254, or 291 ppm ACN for 3 hr. ACN and CEO concentrations were measured in blood, brain, and liver at selected postexposure time points. The dosimetry description accurately simulated the ACN inhalation pharmacokinetic data, providing verification of the parameter estimates. The verified rat dosimetry description for ACN and CEO will be used as the basis for development of a dosimetry description for ACN in people.

Prediction of furan pharmacokinetics from hepatocyte studies: comparison of bioactivation and hepatic dosimetry in rats, mice, and humans.

Furan is a volatile solvent and chemical intermediate that is hepatotoxic and hepatocarcinogenic in rats and mice but is not mutagenic or DNA-reactive. Furan hepatotoxicity requires cytochrome P450 2E1 bioactivation to cis-2-butene-1,4-dial. We have previously shown that furan biotransformation kinetics determined with freshly isolated rat hepatocytes in vitro accurately predict furan pharmacokinetics in vivo [Kedderis et al. (1993) Toxicol. Appl. Pharmacol. 123, 274], suggesting that furan biotransformation kinetics determined with freshly isolated mouse or human hepatocytes can be used to develop species-specific pharmacokinetic models. Hepatocytes from male B6C3F1 mice or human accident victims (n = 3) were incubated with furan vapors to determine the kinetic parameters for furan bioactivation and compared to our previous data for rat hepatocytes. Isolated hepatocytes from all three species rapidly metabolized furan (Vmax of 48 nmol/hr/10(6) mouse hepatocytes, 19-44 nmol/hr/10(6) human hepatocytes, and 18 nmol/hr/10(6) rat hepatocytes) with high affinity (KM ranging from 0.4 to 3.3 microM). The hepatocyte kinetic data and physiological parameters from the literature were used to develop dosimetry models for furan in mice and people. The hepatocyte Vmax values were extrapolated to whole animals assuming 128 x 10(6) hepatocytes/g rodent liver and 137 x 10(6) hepatocytes/g human liver. Simulations of inhalation exposure to 10 ppm furan for 4 hr indicated that the absorbed dose (mg/kg), and consequently the liver dose of cis-2-butene-1,4-dial, was approximately 3- and 10-fold less in humans than in rats or mice, respectively. These results indicate that the target organ concentration, rather than the exposure concentration, is most appropriate for interspecies comparison of dose. The initial rates of furan oxidation in rat, mouse, and human liver were approximately 13-, 24-, and 37-fold greater than the respective rates of blood flow delivery of furan to the liver after 4-hr exposures to < or = 300 ppm. One important consequence of blood flow limitation of furan bioactivation is that the amount of toxic metabolite formed in the liver will be unaffected by increases in Vmax due to the induction of cytochrome P450 2E1. Therefore, the interindividual variations observed in cytochrome P450 2E1 activity among human populations would not be expected to have a significant effect on the extent of furan bioactivation in people. These considerations may be important for human cancer risk assessments of other rapidly metabolized rodent carcinogens.

Furan-induced cytolethality in isolated rat hepatocytes: correspondence with in vivo dosimetry.

Furan, a rodent hepatotoxicant and hepatocarcinogen, produced incubation time- and concentration-dependent decreases in the glutathione (GSH) content and viability of freshly isolated F-344 rat hepatocytes in vitro. Since furan itself did not significantly react with GSH, these data indicate the formation of a reactive metabolite of furan in hepatocyte suspensions. Treatment of the hepatocyte suspensions with the cytochrome P450 inhibitor 1-phenylimidazole delayed GSH depletion but did not alter furan-induced (4 to 12 mM) cytolethality. The furan concentrations required to produce measurable hepatocyte cytolethality in vitro within 6 hr (4 to 12 mM) were several orders of magnitude greater than the predicted maximal liver concentrations of furan in vivo following hepatotoxic doses. In order to study the mechanisms involved in the cytolethality of furan toward hepatocytes in vitro at concentrations relevant to hepatotoxicity in vivo, a hepatocyte suspension/culture system was developed that utilized furan concentrations and incubation times similar to hepatic dosimetry in vivo. Freshly isolated rat hepatocytes in suspension (in Williams' Medium E) were incubated with furan (2 to 100 microM) for 1-4 hr and placed in culture, and viability was determined after 24 hr by lactate dehydrogenase release. Furan produced cytolethality (5 to 70%) and modest GSH depletion in an incubation time- and concentration-dependent manner. Both GSH depletion and cytolethality induced by furan were prevented by 1-phenylimidazole and enhanced by acetone pretreatment of the rats. These data show that oxidation of furan by cytochrome P450 is required for GSH depletion and cytolethality, indicating that a reactive metabolite is involved in cell death. The results of this study underscore the importance of using in vivo toxicant concentrations and exposure times for in vitro mechanistic studies of chemically induced cytolethality.

Kinetic analysis of furan biotransformation by F-344 rats in vivo and in vitro.

Furan is both hepatotoxic and hepatocarcinogenic in rats. The kinetics of furan biotransformation by male F-344 rats were studied in vivo and in vitro in order to understand target tissue dosimetry. A physiologically based pharmacokinetic (PBPK) model for furan in rats was developed from gas uptake studies using initial furan concentrations of 100, 500, 1050, and 3850 ppm. Tissue partition coefficients for furan were determined in vitro using vial equilibration techniques. Furan gas uptake kinetics in vivo were described by a single saturable process with a Vmax of 27.0 mumol/hr/250 g rat and a KM of 2.0 microM. Furan metabolism in vivo was inhibited by pyrazole. The furan PBPK model adequately simulated blood and liver furan concentrations following 4-hr inhalation exposures to 52, 107, and 208 ppm furan. The biotransformation of furan was studied in freshly isolated rat hepatocytes in vitro and compared to biotransformation in vivo. Furan biotransformation by isolated rat hepatocytes exhibited a KM of 0.4 microM and a Vmax of 0.018 mumol/hr/10(6) cells. Inhibition and induction studies indicated that cytochrome P450 was the catalyst of furan oxidation. Acetone pretreatment of the rats produced a five-fold increase in the rate of the hepatocyte oxidation of furan, suggesting an important role for cytochrome P450 2E1. The Vmax determined in hepatocytes in vitro extrapolated to 23.0 mumol/hr/250 g rat, assuming 128 x 10(6) hepatocytes/g liver. Incorporation of the in vitro hepatocyte kinetic parameters into the PBPK model for furan accurately simulated in vivo pharmacokinetics. These results suggest that freshly isolated hepatocytes are a valuable in vitro system for predicting chemical pharmacokinetics in vivo.

Rodent tissue distribution of 2-cyanoethylene oxide, the epoxide metabolite of acrylonitrile.

The direct acting mutagen 2-cyanoethylene oxide (CEO), formed in the liver by oxidation of acrylonitrile (ACN), is thought to mediate the extrahepatic carcinogenic effects of ACN in rats. This study determined the tissue distribution of CEO (3 mg/kg p.o.) in F-344 rats and B6C3F1 mice. Radioactivity from [2,3-14C]CEO was widely distributed in the major organs of rodents by 2 h and decreased by 71% to 90% within 24 h, demonstrating that there was no preferential tissue uptake or retention of CEO. CEO was detected in rodent blood and brain 5-10 min after an oral dose of ACN (10 mg/kg), demonstrating that this mutagenic epoxide metabolite circulates to extrahepatic target organs following ACN administration.

Dose-dependent urinary excretion of acrylonitrile metabolites by rats and mice.

The dose dependence of the urinary excretion of acrylonitrile (ACN) metabolites was studied after oral administration of [2,3-14C]ACN to male F-344 rats (0.09 to 28.8 mg/kg) and male B6C3F1 mice (0.09 to 10.0 mg/kg). Urine was the major route of excretion of ACN metabolites (77 to 104% of the dose), with less than 8% of the dose excreted in the feces. Reverse-phase HPLC analysis of urine from treated animals indicated five major components (1 through 5 in order of elution) that accounted for 75 to 100% of the total urinary radioactivity. Component 4 was observed in the urine of ACN-treated mice but was only present in trace amounts in the urine of ACN-treated rats. Components 1, 2, and 3 were present in the urine of animals administered [2,3-14C]cyanoethylene oxide (CEO), indicating that these components were derived from the epoxide metabolite of ACN. The ACN urinary metabolites were isolated by HPLC and identified by chromatographic and mass spectral analysis. Component 5 was N-acetyl-S-(2-cyanoethyl)cysteine and component 4 was S-(2-cyanoethyl)thioacetic acid, both derived from the glutathione (GSH) conjugate of ACN. Component 3 contained N-acetyl-S-(2-hydroxyethyl)cysteine, N-acetyl-S-(carboxymethyl)cysteine, and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine. Component 2 was thiodiglycolic acid. These urinary metabolites are derived from catabolism of the GSH conjugates of CEO. The polar component 1 was not identified. These results demonstrate that GSH conjugation is the major disposition pathway of ACN. The excretion of metabolites derived from CEO was an approximately linear function of dose in both species, whereas the excretion of N-acetyl-S-(2-cyanoethyl)cysteine increased nonlinearly with dose. This nonlinearity indicates the presence of a saturable pathway competing with glutathione for ACN, most likely the cytochrome P450-dependent oxidation of ACN. Thiodiglycolic acid was formed 10-fold more in mice than in rats, but this species difference in the oxidative processing of GSH conjugates is probably not of toxicological significance. The ratio of ACN epoxidation to GSH conjugation was 0.50 in rats and 0.67 in mice. This species difference in ACN oxidation could have important toxicological implications, since CEO is believed to mediate the carcinogenic effects of ACN.

Metabolism of chloronitrobenzenes by isolated rat hepatocytes.

The metabolism of radiolabeled monochloronitrobenzene isomers was compared in isolated hepatocytes and hepatic subcellular fractions from male Fischer-344 rats. 2-Chloronitrobenezene was converted by isolated hepatocytes to 2-chloroaniline, 2-chloroaniline-N-glucuronide, and S-(2-nitrophenyl)glutathione in approximately equal quantities (13-19% of the added substrate in 90 min). The major metabolite formed from 3-chloronitrobenzene by isolated hepatocytes was 3-chloroaniline (31% of the added substrate in 90 min). Smaller amounts of 3-chloroaniline-N-glucuronide and 3-chloroacetanilide were formed (7 and 17% of the added 3-chloronitrobenzene, respectively, in 90 min). 4-Chloronitrobenzene was metabolized to 4-chloroacetanilide, 4-chloroaniline, and S-(4-nitrophenyl)glutathione in approximately equal amounts (10-15% of the added substrate in 90 min). Studies with hepatic microsomes showed that reduction of the chloronitrobenzenes to chloroanilines was inhibited by SKF 525-A, metyrapone, and carbon monoxide, suggesting that cytochrome P-450 played a role in the reaction. Thus, the major difference in the in vitro hepatic metabolism of the three isomers of chloronitrobenzene is the failure of 3-chloronitrobenzene to be converted to a glutathione conjugate.