Safety of a topically applied metaflumizone spot-on formulation for flea control in cats and kittens.

Four laboratory studies were conducted in cats of various ages to evaluate the safety of a novel low-volume topical spot-on containing 20% metaflumizone (ProMeris for Cats, Fort Dodge Animal Health, Overland Park, KS) when used in cats according to the recommended minimum dosage of 40mg metaflumizonekg(-1) delivered via fixed volume doses of 0.8ml for cats 4.0kg. Study parameters included body weight, food consumption, clinical, physical and neurological examinations, and clinical pathology including complete hematology, coagulation, clinical chemistry and urinalysis. Exaggerated and repeated topical applications of metaflumizone at 1x, 3x and 5x the proposed recommended dose in adult cats and kittens 8 weeks of age had no effect on mortality, body weight, food consumption, clinical, physical or neurological examinations, or clinical pathology parameters. Transient salivation was sporadically noted following some, but not all treatment applications. It occurred and resolved within minutes of treatment application in all groups, including cats treated with placebo. Consequently, it was not considered a direct result of treatment with the active ingredient, metaflumizone. Cats orally administered 10% of the recommended topical dose exhibited considerable avoidance behaviors including spitting, head shaking, and salivation. Therefore, voluntary oral exposure is unlikely. No other adverse signs were observed. Repeated use of metaflumizone caused no adverse health effects when administered at 5x the recommended dose and is safe when used as directed, even on kittens as young as 8 weeks of age.

Safety of a topically applied spot-on formulation of metaflumizone plus amitraz for flea and tick control in dogs.

Four laboratory studies were conducted in Beagle dogs to evaluate the safety of a novel ectoparasiticide combination of metaflumizone plus amitraz (ProMeris/ProMeris Duo for Dogs, Fort Dodge Animal Health, Overland Park, KS) when applied according to the recommended dosage of >/=20mgmetaflumizonekg(-1) plus >/=20mgamitrazkg(-1), at exaggerated and repeated dosages, and if accidentally orally ingested. Parameters evaluated included body weight, food consumption, clinical, physical and neurological examinations, clinical pathology and gross and microscopic pathology. Exaggerated and repeated topical treatment with metaflumizone plus amitraz administered at 1x, 3x and 5x the recommended dose had no effect on clinical findings, heart rates, body weight, food consumption, physical/neurological examinations, macroscopic and microscopic pathology. Very slight, transient, clinically insignificant increases in serum urea nitrogen were noted in some dogs treated at all dose rates tested. This effect was not persistent, was not dose-responsive, nor aggravated by repeated applications and was not associated with a corresponding increase in creatinine or renal pathology. Therefore, these increases in urea nitrogen were suspected to be of non-renal origin and were not considered toxicologically significant. Exaggerated doses (3x and 5x) caused very mild, transient hyperglycemia, most notably in some adult females. Transient and inconsistently noted mild increases in leukocytes, neutrophils and monocytes were observed in some 3x and 5x treated dogs at some intervals. None of the effects noted were aggravated by repeated administration. When 10% of the recommended topical dose was orally administered to mimic exposure due to licking the application, avoidance behaviors including spitting, head shaking, and salivation were noted immediately in all animals. Consequently, voluntary oral ingestion is considered unlikely. Transient decreased activity, slightly reduced body temperature and pale oral mucous membranes were noted in some animals beginning 1-2h posttreatment. Ataxia, resolving within 4h posttreatment, was noted in one female. Oral administration had no effect on clinical pathology. Results from these four studies indicate repeated use of metaflumizone plus amitraz causes no adverse health effects when used as recommended in dogs as young as 8 weeks of age.

cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression.

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.

Constitutive expression of class 3 aldehyde dehydrogenase in cultured rat corneal epithelium.

Mammalian Class 3 aldehyde dehydrogenase (ALDH) is normally associated with neoplastic transformation or xenobiotic induction by aromatic hydrocarbons in liver. However, Class 3 ALDH is constitutively expressed at it's highest specific activity in corneal epithelium. Tissue-specific, differential gene expression is often controlled by alternative, independent molecular pathways. We report here the development of an in vitro corneal epithelium culture system that retains constitutive high expression of the ALDH3 gene. This model system was used to establish, by enzymatic assays, Western and Northern analyses, histochemical and immunocytochemical staining, and 5'3' RACE methodologies that constitutive and xenobiotic induction of Class 3 ALDHs occurs from a single gene. Our results also provide a plausible explanation for the very high Class 3 ALDH activity in mammalian cornea, as the primary mechanism of oxidation of lipid peroxidation-derived aldehydes. Further studies with corneal epithelium suggest the presence of additional mechanisms, other than Ah-receptor-mediated, by which the ALDH3 gene can be differentially regulated in a tissue-specific manner.