Investigation of functional cytochrome P450 4A enzymes in liver and kidney of pigs, cats, tree shrews, and dogs in comparison with the metabolic capacity of human P450 4A11.

Pigs are sometimes utilized in preclinical drug metabolism studies, with growing interest, and so their drug-metabolizing enzymes, including the cytochromes P450 (P450 or CYP; EC 1.14.14.1), need to be examined. In the present study, novel CYP4A cDNAs were isolated and characterized, namely, pig CYP4A23 and CYP4A90; cat CYP4A37 and CYP4A106; and tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g. For comparison, the following known CYP4A cDNAs were also analyzed: pig CYP4A21 and dog CYP4A37, CYP4A38, and CYP4A39. These CYP4A cDNAs all contained open reading frames of 504-513 amino acids and had high amino acid sequence identity (74-80%) with human CYP4As. Phylogenetic analysis of amino acid sequences revealed that these CYP4As were clustered in each species. All CYP4A genes contained 12 coding exons and formed a gene cluster in the corresponding genomic regions. A range of tissue types were analyzed, and these CYP4A mRNAs were preferentially expressed in liver and/or kidney, except for pig CYP4A90, which showed preferential expression in lung and duodenum. CYP4A enzymes, heterologously expressed in Escherichia coli, preferentially catalyzed lauric acid 12-hydroxylation and arachidonic acid 20-hydroxylation, just as human CYP4A11 does, with the same regioselectivity, i.e., at the ω-position of fatty acids. These results imply that dog, cat, pig, and tree shrew CYP4As have functional characteristics similar to those of human CYP4A11, with minor differences in lauric acid 12-hydroxylation. Significance Statement Cytochrome P450 (P450, CYP) 4As are important P450s in human biological processes because of their fatty acid-metabolizing ability. Pig CYP4A21, CYP4A23, and CYP4A90; cat CYP4A37 and CYP4A106; tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g; and dog CYP4A37, CYP4A38, and CYP4A39 cDNAs were isolated and analyzed. These CYP4A cDNAs shared relatively high sequence identities with human CYP4A11 and CYP4A22. Pig, cat, tree shrew, and dog CYP4As in the liver and kidneys are likely to catalyze the ω-hydroxylation of fatty acids.

Quantification of the tissue distribution and accumulation of the neonicotinoid pesticide clothianidin and its metabolites in maternal and fetal mice.

Neonicotinoids (NNs) are commonly used pesticides that have a selective agonistic action on insect nicotinic acetylcholine receptors. Recent evidence has shown that NNs have adverse effects in the next generation of mammals, but it remains unclear how NNs transferred from dams to fetuses are distributed and accumulated in fetal tissues. Here, we aimed to clarify the tissue distribution and accumulation properties of the NN clothianidin (CLO) and its 6 metabolites in 7 tissues and blood in both dams and fetuses of mice administered CLO for a single day or for 9 consecutive days. The results showed that the total concentrations of CLO-related compounds in the brain and kidney were higher in fetuses than in dams, whereas in the liver, heart, and blood they were lower in fetuses. The multi-day administration increased the total levels in heart and blood only in the fetuses of the single administration group. In addition, dimethyl metabolites of CLO showed fetus/dam ratios >1 in some tissues, suggesting that fetuses have higher accumulation property and are thus at higher risks of exposure to CLO-related compounds than dams. These findings revealed differences in the tissue-specific distribution patterns of CLO and its metabolites between dams and fetuses, providing new insights into the assessment of the developmental toxicity of NNs.

Sensitivity assessment of diphacinone by pharmacokinetic analysis in invasive black rats in the Bonin (Ogasawara) Archipelago, Japan.

The Bonin Archipelago is a United Nations Educational, Scientific and Cultural Organization's World Natural Heritage Site in Japan with a unique ecosystem; however, the invasive rodents preying on endemic species have been a significant concern. The anticoagulant rodenticide, diphacinone, sprayed by the Ministry of the Environment, has succeeded; however, its repeated use leads to rodenticide resistance. This study evaluated the sensitivity by in vivo pharmacokinetics/pharmacodynamics (PK/PD) analysis and physiologically-based pharmacokinetic modeling to diphacinone in black rats (Rattus rattus) captured on the Bonin Archipelago in February 2022. The Bonin rats exhibited prolonged coagulation time after diphacinone administration. They recovered earlier than susceptible black rats, indicating that Bonin rats were less susceptible, though there were no genetic mutations in Vkorc1, the target enzyme of diphacinone. After the administration of diphacinone, hepatic expression levels of Fsp1, identified as the vitamin K reductase, was decreased, however, the Bonin rats exhibited the most minor suppression. The PK analysis showed that the excretion capacity of the Bonin rats was lower than that of the resistant black rats. In the PBPK modeling, the resistant black rats showed higher clearance than the Bonin and susceptible black rats due to high hepatic metabolic capacity. The Bonin rats demonstrated slow absorption and relatively low clearance. This study highlighted the reduced rodenticide-sensitive tendency of wild black rats in the Bonin Archipelago at an in vivo phenotype level. At the same time, they do not have known rodenticide resistance mechanisms, such as hepatic metabolic enhancement or Vkorc1 mutations. It is crucial to monitor the biological levels to evaluate rodenticide sensitivity accurately.

Insights on hazardous metal bioaccessibility, and groundwater impacted by Zn residues from a legacy mine and risk evaluation of adjacent soils.

This study explored the legacy impact of Zinc plant residues (ZPRs) in Kabwe, Zambia, on the environment and human health, particularly in light of the town's reputation for Pb pollution. ZPRs solid samples and groundwater within and around ZPRs zone were collected from the legacy mine, along with soils in a 10 km radius from the mine site. Bioaccessible fractions of Pb and Zn were elucidated by Japanese leaching test (JLT) and simple bioaccessibility extraction test (SBET). Cationic speciation of Pb and Zn from inhalable and ingestible ZPRs particles was investigated via sequential extraction. Groundwater in the ZPRs area showed higher Zn levels (1490 mg/L) compared to Pb (1.7 mg/L). Elevated Zn concentration were facilitated by the presence of soluble Zn sulfates while Pb was constrained due to its precipitation as anglesite. Groundwater sampled outside the ZPRs area was within the Zambia regulatory limits (< 0.5 mg/L for Pb and < 1 mg/L for Zn). Inhalation exposure to < 30 µm dust particles from ZPRs and soils near the mine indicated negligible risk, with < 3% of bioaccessible Pb in artificial lysosomal fluid. Meanwhile, oral intake of ZPRs particles < 250 µm revealed elevated bioaccessible fractions (36% for Pb and 70% for Zn). ZPRs cationic speciation of ingestible particles < 30 µm, 30-75 µm, 75-150 µm and 150-250 µm indicated that the bioaccessible Pb predominantly emanated from labile Pb fractions under gastric conditions with pH < 1. This was due to the dissolution of Pb associated with the exchangeable phase, carbonates and iron/manganese oxides; however, only exchangeable/carbonate Pb was bioaccessible at pH < 2. Hazard quotients indicated increased risks of Pb intoxication through the ingestion of ZPRs and soils near the legacy mine, with higher risks observed in children, emphasizing the need to remediate legacy mine wastes to reduce health risks and protect groundwater through monitoring in mining-affected regions.

A stable isotope analysis of the dietary patterns of the aquatic apex predator, the African tigerfish (Hydrocynus vittatus).

Stable isotope analyses, specifically δ13 C and δ15 N, are useful tools increasingly used to understand ecosystem function, food web structures, and consumer diets. Although the iconic tigerfish Hydrocynus vittatus is regarded as an apex predator in southern African freshwater systems, little information is available regarding their feeding behavior and how this may change with growth or differ between ecosystems, with most information stemming from stomach content analyses (SCA). The aim of the present study was to address this lack of information through a baseline study of the diet of large and small tigerfish in various lentic and lotic ecosystems in South Africa using stable isotope methods. Fish and various food web components and food sources were collected from two river and two lake ecosystems in South Africa. The δ13 C and δ15 N values for all samples were determined and multivariate analyses and Bayesian analytical techniques applied to determine the feeding ecology of H. vittatus and how this may differ with size and habitat type. Analyses revealed a substantial difference in the type and abundance of food sources contributing to the diet of H. vittatus between ecosystems, most prominently between the lotic systems, where less dietary specialization was observed, and lentic systems where more specialization was observed. Furthermore, there was a distinct difference in diet between small and large tigerfish, especially in the lotic system, indicating an ontogenetic diet shift as tigerfish grow and further supporting previous SCA studies. This is the first study of its kind on the African continent for H. vittatus and the findings illustrate the value of stable isotope analysis in providing in-depth information into the feeding ecology of consumers and how this may differ between size classes and habitat types.

Cytochrome P450 2J Genes Are Expressed in Dogs, Cats, and Pigs, and Encode Functional Drug-Metabolizing Enzymes.

Cytochrome P450s (P450s) have been identified and analyzed in dogs and pigs, species that are often used in preclinical drug studies. Moreover, P450s are clinically important for drug therapy not only in humans, but also in species under veterinary care, including dogs and cats. In the present study, seven P450s homologous to human CYP2J2, namely, dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J35, CYP2J91, and CYP2J93, were newly identified and characterized, along with pig CYP2J34 previously identified. The cDNAs of these CYP2Js contain open reading frames of 502 amino acids, except for CYP2J35 (498 amino acids), and share high sequence identity (77%-80%) with human CYP2J2. Phylogenetic analysis revealed that dog and cat CYP2J2 were closely related, whereas pig CYP2Js formed a cluster. All seven CYP2J genes contain nine coding exons and are located in corresponding genomic regions, with the pig CYP2J genes forming a gene cluster. These CYP2J2 mRNAs were predominantly expressed in the small intestine with additional expression in the kidney and brain for dog CYP2J2 and pig CYP2J91 mRNAs, respectively. All seven CYP2Js metabolized human CYP2J2 substrates terfenadine, ebastine, and astemizole, indicating that they are functional enzymes. Dog CYP2J2 and pig CYP2J34 and CYP2J35 efficiently catalyzed ebastine primary hydroxylation and secondary carebastine formation at low substrate concentrations, just as human CYP2J2 does. Velocity-versus-substate plots exhibited sigmoidal relationships for dog CYP2J2, cat CYP2J2, and pig CYP2J33, indicating allosteric interactions. These results suggest that dog, cat, and pig CYP2Js have similar functional characteristics to human CYP2J2, with slight differences in ebastine and astemizole oxidations. SIGNIFICANCE STATEMENT: Dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J34, CYP2J35, CYP2J91, and CYP2J93, homologous to human CYP2J2, were identified and characterized by sequence, phylogenetic, and genomic structure analyses. Intestinal expression patterns of CYP2J mRNAs were characteristic in dogs, cats, and pigs. Dog, cat, and pig CYP2Js likely play roles as drug-metabolizing enzymes in the small intestine, similar to human CYP2J2.

Sex-specific behavioral effects of acute exposure to the neonicotinoid clothianidin in mice.

Although neonicotinoids are among the major classes of pesticides that affect mammalian nervous systems, little is known about sex differences in their effects. This study aimed to examine whether the neurobehavioral effects of a neonicotinoid, clothianidin (CLO), differed between sexes. Male and female C57BL/6N mice were orally administered CLO (5 or 50 mg/kg) at or below the chronic no-observed-adverse-effect-level (NOAEL) and subjected to behavioral tests of emotional and learning functions. Changes in neuroactivity in several brain regions and the concentrations of CLO and its metabolites in blood and urine were measured. Acute CLO exposure caused sex-related behavioral effects; decreases in locomotor activities and elevation of anxiety-like behaviors were more apparent in males than in females. In addition, male-specific impairment of short- and long-term learning memory by CLO exposure was observed in both the novel recognition test and the Barnes maze test. Male-dominant increases in the number of c-fos positive cells were observed in the paraventricular thalamic nucleus in the thalamus and in the dentate gyrus in the hippocampus, which are related to the stress response and learning function, respectively. The concentrations of CLO and most metabolites in blood and urine were higher in males. These results support the notion that male mice are more vulnerable than females to the neurobehavioral effects of CLO and provide novel insights into the risk assessment of neonicotinoids in mammalian neuronal function.

Geospatial approach to investigate spatial clustering and hotspots of blood lead levels in children within Kabwe, Zambia.

BACKGROUND: Communities around Kabwe, Zambia are exposed to lead due to deposits from an old lead (Pb) and zinc (Zn) mining site. Children are particularly more vulnerable than adults, presenting with greatest risk of health complications. They have increased oral uptake due to their hand to mouth activities. Spatial analysis of childhood lead exposure is useful in identifying specific areas with highest risk of pollution. The objective of the current study was to use a geospatial approach to investigate spatial clustering and hotspots of blood lead levels in children within Kabwe. METHODS: We analysed existing data on blood lead levels (BLL) for 362 children below the age of 15 from Kabwe town. We used spatial autocorrelation methods involving the global Moran's I and local Getis-Ord Gi*statistic in ArcMap 10.5.1, to test for spatial dependency among the blood lead levels in children using the household geolocations. RESULTS: BLL in children from Kabwe are spatially autocorrelated with a Moran's Index of 0.62 (p < 0.001). We found distinct hotspots (mean 51.9 µg/dL) in communities close to the old lead and zinc-mining site, lying on its western side. Whereas coldspots (mean 7 µg/dL) where observed in areas distant to the mine and traced on the eastern side. This pattern suggests a possible association between observed BLL and distance from the abandoned lead and zinc mine, and prevailing winds. CONCLUSION: Using geocoded data for households, we found clustering of childhood blood lead and identified distinct hotspot areas with high lead levels for Kabwe town. The geospatial approach used is especially valuable in resource-constrained settings like Zambia, where the precise identification of high risk locations allows for the initiation of targeted remedial and treatment programs.

Toxicokinetic analysis of the anticoagulant rodenticides warfarin & diphacinone in Egyptian fruit bats (Rousettus aegyptiacus) as a comparative sensitivity assessment for Bonin fruit bats (Pteropus pselaphon).

Anticoagulant rodenticides have been widely used to eliminate wild rodents, which as invasive species on remote islands can disturb ecosystems. Since rodenticides can cause wildlife poisoning, it is necessary to evaluate the sensitivity of local mammals and birds to the poisons to ensure the rodenticides are used effectively. The Bonin Islands are an archipelago located 1000 km southeast of the Japanese mainland and are famous for the unique ecosystems. Here the first-generation anticoagulant rodenticide diphacinone has been used against introduced black rats (Rattus rattus). The only land mammal native to the archipelago is the Bonin fruit bat (Pteropus pselaphon), but little is known regarding its sensitivity to rodenticides. In this study, the Egyptian fruit bats (Rousettus aegyptiacus) was used as a model animal for in vivo pharmacokinetics and pharmacodynamics analysis and in vitro enzyme kinetics using their hepatic microsomal fractions. The structure of vitamin K epoxide reductase (VKORC1), the target protein of the rodenticide in the Bonin fruit bat, was predicted from its genome and its binding affinity to rodenticides was evaluated. The Egyptian fruit bats excreted diphacinone slowly and showed similar sensitivity to rats. In contrast, they excreted warfarin, another first-generation rodenticide, faster than rats and recovered from the toxic effect faster. An in silico binding study also indicated that the VKORC1 of fruit bats is relatively tolerant to warfarin, but binds strongly to diphacinone. These results suggest that even chemicals with the same mode of action display different sensitivities in different species: fruit bat species are relatively resistant to warfarin, but vulnerable to diphacinone.

The VKORC1 ER-luminal loop mutation (Leu76Pro) leads to a significant resistance to warfarin in black rats (Rattus rattus).

Well-known 4-hydroxycoumarin derivatives, such as warfarin, act as inhibitors of the vitamin K epoxide reductase (VKOR) and are used as anticoagulants. Mutations of the VKOR enzyme can lead to resistance to those compounds. This has been a problem in using them as medicine or rodenticide. Most of these mutations lie in the vicinity of potential warfarin-binding sites within the ER-luminal loop structure (Lys30, Phe55) and the transmembrane helix (Tyr138). However, a VKOR mutation found in Tokyo in warfarin-resistant rats does not follow that pattern (Leu76Pro), and its effect on VKOR function and structure remains unclear. We conducted both in vitro kinetic analyses and in silico docking studies to characterize the VKOR mutant. On the one hand, resistant rats (R-rats) showed a 37.5-fold increased IC50 value to warfarin when compared to susceptible rats (S-rats); on the other hand, R-rats showed a 16.5-fold lower basal VKOR activity (Vmax/Km). Docking calculations exhibited that the mutated VKOR of R-rats has a decreased affinity for warfarin. Molecular dynamics simulations further revealed that VKOR-associated warfarin was more exposed to solvents in R-rats and key interactions between Lys30, Phe55, and warfarin were less favored. This study concludes that a single mutation of VKOR at position 76 leads to a significant resistance to warfarin by modifying the types and numbers of intermolecular interactions between the two.

The effects of fipronil on emotional and cognitive behaviors in mammals.

Fipronil is a phenylpyrazole insecticide that is widely used as a pesticide and a veterinary drug, although studies suggest that it could be toxic to mammals. The objectives of this study were to examine the pharmacokinetic profile of fipronil in mice, dogs, and cats, and to evaluate its effects on emotional and cognitive behaviors of dogs and cats using the data obtained from mice. The assessment of in vivo kinetics of fipronil was conducted in mice and dogs. We also performed behavioral tests (elevated plus-maze and Y-maze) and measured the levels of neurotransmitters in mice exposed to fipronil. In addition, the in vitro metabolism of fipronil were evaluated using liver microsomes of rats, mice, dogs, and cats. The results revealed that fipronil is distributed throughout the body (blood, brain, adipose tissue, and liver) of mice after dermal application. It was metabolized to fipronil sulfone primarily in the liver. The data on kinetics show that both fipronil and fipronil sulfone have a longer half-life in dogs and cats than in mice. The behavioral tests indicated that fipronil and fipronil sulfone could affect emotional and cognitive behaviors and alter the levels of neurotransmitters (dopamine in the striatum and serotonin in the hippocampus) in mice. Furthermore, we found that dogs and cats have a low ability to metabolize fipronil than mice and rats. However, further comprehensive studies are needed to determine whether fipronil affects the emotional and cognitive behaviors when administered to dogs and cats. To the best of our knowledge, this is the first study to examine the pharmacokinetic data and verify the effects of fipronil on emotional and cognitive behaviors of dogs and cats using the data obtained from mice.

Land Use in Habitats Affects Metal Concentrations in Wild Lizards Around a Former Lead Mining Site.

We investigated the potential effects of different land use and other environmental factors on animals living in a contaminated environment. The study site in Kabwe, Zambia, is currently undergoing urban expansion, while lead contamination from former mining activities is still prevalent. We focused on a habitat generalist lizards (Trachylepis wahlbergii). The livers, lungs, blood, and stomach contents of 224 lizards were analyzed for their lead, zinc, cadmium, copper, nickel, and arsenic concentrations. Habitat types were categorized based on vegetation data obtained from satellite images. Multiple regression analysis revealed that land use categories of habitats and three other factors significantly affected lead concentrations in the lizards. Further investigation suggested that the lead concentrations in lizards living in bare fields were higher than expected based on the distance from the contaminant source, while those in lizards living in green fields were lower than expected. In addition, the lead concentration of lungs was higher than that of the liver in 19% of the lizards, implying direct exposure to lead via dust inhalation besides digestive exposure. Since vegetation reduces the production of dust from surface soil, it is plausible that dust from the mine is one of the contamination sources and that vegetation can reduce exposure to this.

Blood lead levels and aberrant DNA methylation of the ALAD and p16 gene promoters in children exposed to environmental-lead.

BACKGROUND: Lead (Pb) is a well-known toxic heavy metal which can have serious public health hazards. As of today, there is no safe threshold for Pb exposure, especially for children. Lead exposure has been associated with adverse health outcomes involving epigenetic mechanisms, such as aberrant DNA methylation. The objective of the present study was to elucidate the associations between blood lead levels (BLLs) and gene-specific promoter DNA methylation status in environmental Pb-exposed children from Kabwe, Zambia. METHODS: A cross-sectional study was conducted using 2 to 10-year-old children from high Pb exposed area (N = 102) and low Pb exposed area (N = 38). We measured BLLs using a LeadCare II analyzer and investigated the methylation status of the ALAD and p16 gene promoters by methylation-specific PCR. RESULTS: The mean BLLs were 23.7 µg/dL and 7.9 µg/dL in high Pb exposed and low Pb exposed children, respectively. Pb exposure was correlated with increased methylation of the ALAD and p16 genes. The promoter methylation rates of ALAD and p16 in high Pb exposed children were 84.3% and 67.7%, and 42.1% and 44.7% in low Pb exposed children, respectively. Significantly increased methylation was found in both genes in high Pb exposed children compared with low Pb exposed children (p < 0.05). Children with methylated ALAD and p16 genes showed an increased risk of Pb poisoning (odd ratio >1) compared to the unmethylated status. CONCLUSIONS: This study for the first time tries to correlate promoter methylation status of the ALAD and p16 genes in environmental Pb-exposed children from Kabwe, Zambia as a representative. The result suggests that Pb exposure increases aberrations in ALAD and p16 gene methylation, which may be involved in the mechanism of Pb toxicity.

Accumulation patterns and risk assessment of metals and metalloid in muscle and offal of free-range chickens, cattle and goat in Benin City, Nigeria.

The use of free range animals for monitoring environmental health offers opportunities to detect exposure and assess the toxicological effects of pollutants in terrestrial ecosystems. Potential human health risk of dietary intake of metals and metalloid via consumption of offal and muscle of free range chicken, cattle and goats by the urban population in Benin City was evaluated. Muscle, gizzard, liver and kidney samples were analyzed for Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Cd, and Pb concentrations using inductively coupled plasma mass spectrometer (ICP-MS) while Hg was determined using Hg analyzer. Mean concentrations of metals (mg/kg ww) varied significantly depending upon the tissues and animal species. Human health risk estimations for children and adults showed estimated daily intake (EDI) values of tissues below oral reference dose (RfD) threshold for non essential metals Cd, As, Pb and Hg thus strongly indicating no possible health risk via consumption of animal based food. Calculated Hazard quotient (THQ) was less than 1 (< 1) for all the metals analyzed for both adult and children. However, Cd and As had the highest value of THQ suggestive of possible health risk associated with continuous consumption of Cd and As contaminated animal based foods. Hazard Index (HI) for additive effect of metals was higher in chicken liver and gizzard for children and chicken liver for adults. Thus, HI indicated that chicken liver and gizzard may contribute significantly to adult and children dietary exposure to heavy metals. Principal component analysis (PCA) showed a clear species difference in metal accumulation between chickens and the ruminants. This study provides baseline data for future studies and also valuable evidence of anthropogenic impacts necessary to initiate national and international policies for control of heavy metal and metalloid content in food items.

Linking organochlorine exposure to biomarker response patterns in Anurans: a case study of Müller's clawed frog (Xenopus muelleri) from a tropical malaria vector control region.

Organochlorine pesticides are highly persistent in aquatic ecosystems. Amphibians, specifically anurans, play an intricate part in the aquatic food web, and have very permeable skin which makes them prone to bioaccumulation of persistent pollutants. In this study the bioaccumulation of various legacy organochlorine pesticides (OCPs)-including dichlorodiphenyltrichloroethane (DDT), currently used for malaria vector control (MVC)-was assessed along with a set of biomarker responses in Müller's clawed frog Xenopus muelleri collected from the lower Phongolo River floodplain in South Africa. Possible relationships between bioaccumulation and biomarkers (of exposure, oxidative stress biomarkers, and cellular energy allocation) alongside their temporal changes were investigated. The OCP concentrations showed a significant increase over time for the duration of the study. The increase correlated negatively with rainfall from the region. DDT levels were well below expected effects levels with p,p-DDE being the main contributing metabolite. The results of this study indicate OCPs actively accumulate at sub-lethal levels in aquatic frogs from the study area, while showing possible relations towards some of the biochemical stress responses measured. Most notable were negative relationships indicated between p,p-DDE and acetylcholinesterase, malondialdehyde, and carbohydrates and protein energy availability. Levels of DDT were not found to be significantly higher than other legacy pesticides in the frog tissue, although evidence of newly introduced DDT in the frog tissue was found. Further investigation about sub-lethal effects of these pesticides on anurans is required to gain better insight into their full impact on animal livelihood.

Investigation of hepatic warfarin metabolism activity in rodenticide-resistant black rats (Rattus rattus) in Tokyo by in situ liver perfusion.

Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the Vmax of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450.

Anthropogenic and Naturally Produced Brominated Phenols in Pet Blood and Pet Food in Japan.

Present study determined concentrations and residue patterns of bromophenols (BPhs) in whole blood samples of pet cats and pet dogs collected from veterinary hospitals in Japan. BPhs concentrations were higher in cat blood than in dog blood, with statistically insignificant differences (p = 0.07). Among the congeners, 2,4,6-tribromophenol (TBPh) constituted the majority of BPhs (>90%) detected in both species. Analysis of commercial pet food to estimate exposure routes showed that the most abundant congener in all pet food samples was 2,4,6-TBPh, accounting for >99% of total BPhs. This profile is quite similar to the blood samples of the pets, suggesting that diet might be an important exposure route for BPhs in pets. After incubation in polybrominated diphenyl ether (PBDE) mixtures (BDE-47, BDE-99 and BDE-209), 2,4,5-TBPh was found in dog liver microsomes but not in cat liver microsomes, implying species-specific metabolic capacities for PBDEs. Formation of 2,4,5-TBPh occurred by hydroxylation at the 1' carbon atom of the ether bond of BDE-99 is similar to human study reported previously. Hydroxylated PBDEs were not detected in cats or dogs; therefore, diphenyl ether bond cleavage of PBDEs can also be an important metabolic pathway for BPhs formation in cats and dogs.

Trace Element Contamination in Tissues of Four Bird Species from the Rift Valley Region, Ethiopia.

Concentrations of ten trace elements (Hg, As, Cd, Pb, Co, Cr, Cu, Ni, Se and Zn) were determined in different tissues (liver, kidney, muscle, heart and brain) of African sacred ibis (Threskiornis aethiopicus), Hamerkop (Scopus umbretta), marabou stork (Leptoptilos crumeniferus) and great white pelican (Pelecanus onocrotalus) inhabiting the Ethiopian Rift Valley region. There were differences in trace element patterns among the bird species. Significantly (p < 0.05) higher concentrations of Cd (5.53 µg/g dw ± 2.94) in kidney and Hg (0.75 µg/g ww ± 0.30) in liver were observed in the great white pelican compared to the other species, and liver concentrations of these two elements showed positive correlations with trophic level. Concentrations of toxic elements (As, Cd, Pb and Hg) in liver were below their respective toxicological thresholds, indicating that the data may provide baseline information for future studies.

Organohalogen Compounds in Pet Dog and Cat: Do Pets Biotransform Natural Brominated Products in Food to Harmful Hydroxlated Substances?

There are growing concerns about the increase in hyperthyroidism in pet cats due to exposure to organohalogen contaminants and their hydroxylated metabolites. This study investigated the blood contaminants polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) and their hydroxylated and methoxylated derivatives (OH-PCBs, OH-PBDEs, and MeO-PBDEs), in pet dogs and cats. We also measured the residue levels of these compounds in commercially available pet foods. Chemical analyses of PCBs and OH-PCBs showed that the OH-PCB levels were 1 to 2 orders of magnitude lower in cat and dog food products than in their blood, suggesting that the origin of OH-PCBs in pet dogs and cats is PCBs ingested with their food. The major congeners of OH-/MeO-PBDEs identified in both pet food products and blood were natural products (6OH-/MeO-BDE47 and 2'OH-/MeO-BDE68) from marine organisms. In particular, higher concentrations of 6OH-BDE47 than 2'OH-BDE68 and two MeO-PBDE congeners were observed in the cat blood, although MeO-BDEs were dominant in cat foods, suggesting the efficient biotransformation of 6OH-BDE47 from 6MeO-BDE47 in cats. We performed in vitro demethylation experiments to confirm the biotransformation of MeO-PBDEs to OH-PBDEs using liver microsomes. The results showed that 6MeO-BDE47 and 2'MeO-BDE68 were demethylated to 6OH-BDE47 and 2'OH-BDE68 in both animals, whereas no hydroxylated metabolite from BDE47 was detected. The present study suggests that pet cats are exposed to MeO-PBDEs through cat food products containing fish flavors and that the OH-PBDEs in cat blood are derived from the CYP-dependent demethylation of naturally occurring MeO-PBDE congeners, not from the hydroxylation of PBDEs.

Novel revelation of warfarin resistant mechanism in roof rats (Rattus rattus) using pharmacokinetic/pharmacodynamic analysis.

Roof rats (Rattus rattus) live mainly in human habitats. Heavy use of rodenticides, such as warfarin, has led to the development of drug resistance, making pest control difficult. There have been many reports regarding mutations of vitamin K epoxide reductase (VKOR), the target enzyme of warfarin, in resistant rats. However, it has been suggested there are other mechanisms of warfarin resistance. To confirm these possibilities, closed colonies of warfarin-susceptible roof rats (S) and resistant rats from Tokyo (R) were established, and the pharmacokinetics/pharmacodynamics of warfarin in rats from both colonies was investigated. R rats had low levels of warfarin in serum and high clearance activity. These rats can rapidly metabolize warfarin by hydroxylation. The levels of accumulation in the organs were lower than those of S rats. R rats administered warfarin showed high expression levels of CYP2B, 2C, and 3A, which play roles in warfarin hydroxylation, and may explain the high clearance ability of R rats. The mechanism of warfarin resistance in roof rats from Tokyo involved not only mutation of VKOR but also high clearance ability due to high levels of CYP2B, 2C and 3A expression possibly induced by warfarin.