A premature stop codon in the CYP2C19 gene may explain the unexpected sensitivity of vultures to diclofenac toxicity.

The unintended environmental exposure of vultures to diclofenac has resulted in the deaths of millions of old-world vultures on the Asian subcontinent. While toxicity has been since associated with a long half-life of elimination and zero order metabolism, the actual constraint in biotransformation is yet to be clarified. For this study we evaluated if the evident zero order metabolism could be due to defects in the CYP2C9/2C19 enzyme system. For this, using whole genome sequencing and de-novo transcriptome alignment, the vulture CYP2C19 open reading frame was identified through Splign analysis. The result sequence analysis revealed the presence of a premature stop codon on intron 7 of the identified open reading frame. Even if the stop codon was not present, amino acid residue analysis tended to suggest that the enzyme would be lower in activity than the equivalent human enzyme, with differences present at sites 105, 286 and 289. The defect was also conserved across the eight non-related vultures tested. From these results, we conclude that the sensitivity of the old-world vultures to diclofenac is due to the non-expression of a viable CYP2C19 enzyme system. This is not too dissimilar to the effects seen in certain people with a similar defective enzyme.

Diclofenac toxicity in susceptible bird species results from a combination of reduced glomerular filtration and plasma flow with subsequent renal tubular necrosis.

Diclofenac caused the death of millions of vultures on the Asian subcontinent. Other non-steroidal anti-inflammatory drugs (NSAIDs) have since also been shown to be toxic to vultures with the exception of meloxicam. For this study, we evaluated the effect of diclofenac on renal uric acid transport and glomerulus filtration in an acute toxicity model. In a two-phase study with the same birds, healthy chickens (a validated model species) were treated intravenously with para-amino hippuric acid (PAH) and iohexol (IOH) in combination in phase 1. In phase 2, the same PAH and IOH combination was then combined with diclofenac (10 mg/kg). In both phases, blood and faeces were sequentially collected. In phase 1, the birds showed no signs of ill health. Moreover, PAH, IOH and uric acid clearance was rapid. In phase 2, two chickens showed early signs of hyperuricemia 8 hours after exposure and died approximately 24h later. Necropsy showed classic signs of renal damage and gout. Diclofenac had a rapid plasma half-life of elimination of less than 2 hours indicating that toxicity was likely due to an irreversible destruction of a physiological process. All the birds in phase 2 had decreased uric acid, PAH and IOH clearance in comparison to phase 1. The decrease in PAH clearance was variable between the birds (average of 71%) but was near 98% reduced in the two birds that died. It is concluded that diclofenac alters both renal perfusion and renal plasma flow, with death associated with tubular secretion being reduced to negligible functionality for a prolonged period. This would support previous in vitro findings of early cell death from ROS accumulation. However, further evaluation is needed to elucidate this final step.

The complete mitochondrial genome of Gyps coprotheres (Aves, Accipitridae, Accipitriformes): phylogenetic analysis of mitogenome among raptors.

Three species of Old World vultures on the Asian peninsula are slowly recovering from the lethal consequences of diclofenac. At present the reason for species sensitivity to diclofenac is unknown. Furthermore, it has since been demonstrated that other Old World vultures like the Cape (Gyps coprotheres; CGV) and griffon (G. fulvus) vultures are also susceptible to diclofenac toxicity. Oddly, the New World Turkey vulture (Cathartes aura) and pied crow (Corvus albus) are not susceptible to diclofenac toxicity. As a result of the latter, we postulate an evolutionary link to toxicity. As a first step in understanding the susceptibility to diclofenac toxicity, we use the CGV as a model species for phylogenetic evaluations, by comparing the relatedness of various raptor species known to be susceptible, non-susceptible and suspected by their relationship to the Cape vulture mitogenome. This was achieved by next generation sequencing and assembly. The Cape vulture mitogenome had a genome size of 16,908 bp. The mitogenome phylogenetic analysis indicated a close evolutionary relationship between Old World vultures and other members of the Accipitridae as indicated by bootstrap value of 100% on the phylogenetic trees. Based on this, we postulate that the other species could also be sensitive to the toxic effects of diclofenac. This warrants further investigations.

Percentage of faecal excretion of meloxicam in the Cape vultures (Gyps corprotheres).

Asian Gyps vulture species are gradually recovering from the devastating effect of diclofenac being present in contaminated carcasses. This drug was responsible for the death of over 10 million vultures in India, Nepal and Pakistan. To prevent the extinction of vultures, meloxicam was introduced after the ban of veterinary diclofenac. Meloxicam's safety in vultures was attributed to its short elimination half-life in contrast with diclofenac. The reason for the rapid elimination of meloxicam is yet to be explained. The aim of this study was to evaluate the role of biotransformation in the elimination of meloxicam. Six Cape griffon vultures (Gyps coprotheres) were treated with 2 mg/kg meloxicam intramuscularly for faecal and plasma quantification of meloxicam concentration over time. In the plasma meloxicam was characterised by a half-life, mean residence time, clearance and volume of distribution at steady state of 0.37 ±â€¯0.10 h, 0.90 ±â€¯0.12 h, 0.02 ±â€¯0.00 l/h kg and 0.02 ±â€¯0.00 l/kg respectively (presented as geometric mean). Over the 24 h monitoring period, the total non-metabolised meloxicam in the faeces was 1.35 ±â€¯0.71% of the total concentration in the plasma. Based on the short meloxicam elimination half-life and low cumulative concentration of total faecal meloxicam over a period in excess of 10 half-lives, this study indicates that Cape griffon vultures are efficient metaboliser of meloxicam, which is suggestive of different set of cytochrome enzymes being involved in the metabolism to that for diclofenac in this species. Identification of orthologous human CYP2C9 and CYP3A4 enzyme families in vultures will be an important further step in explaining the differences in the metabolic pathway(s) of meloxicam and diclofenac for the species.

The use of liver slices from the Cape vulture (Gyps coprotheres) to better understand the role of liver toxicity of non-steroidal anti-inflammatory drugs (NSAIDs) in vultures.

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID) was responsible for the death of millions of vultures on the Asian subcontinent, following the consumption of diclofenac contaminated carcasses. The aim of this research was to establish if liver slices could serve as an alternate means of predicting the toxicity of NSAIDs in Gyps vultures. The Cape vulture liver slices was prepared and incubated with four NSAIDs for 6 h. A percent clearance of 1.0 ±â€¯0.253, 0.58 ±â€¯0.153, 0.961 ±â€¯0.312 and 1.242 ±â€¯0.406 (%/h*g) was attained for diclofenac, carprofen, ketoprofen and meloxicam respectively. Both meloxicam and diclofenac exerted toxic effects on the hepatic cells. Protein content indicated that the vulture tissue had lower enzyme levels than expected for an animal of its size. The poor distinction between the ex vivo hepatic percent clearance of meloxicam and diclofenac indicates that liver slices is not an ideal model to investigate NSAIDs toxicity in Cape vulture.