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  • ARA metabolites which are produced through

    2019-09-12

    ARA metabolites, which are produced through cytochrome P450 (CYP450) enzymes influence cardiovascular homeostasis. Human CYP4A11 and CYP1A2 metabolize ARA to 20-hydroxyecostearonic Bestatin hydrochloride (20-HETE) [11], which is a vasoconstrictor [12], platelet activator [13] and naturetic in the kidney [14]. On the other hand, human CYP2C9 and CYP2J2 metabolize ARA to epoxyeicosatrienoic acids (EETs) [11], which are vasodilators and are considered cardioprotective agents [15]. Disturbance of ARA-CYP450 metabolites was associated with cardiovascular diseases, such as hypertension [16], and anticancer doxorubicin induced cardiotoxicity [17]. In addition, Lee et al. [13], found that 20-HETE was elevated 120 fold in the plasma of rofecoxib treated mice, which was associated with decreased bleeding time. Furthermore, it was recently reported that the selective COX2 inhibitors meloxicam and rofecoxib altered the ARA-CYP450 metabolites and elevated 20-HETE levels in the cardiac tissues of treated rats [18].
    Materials and methods
    Results After 14 days of NSAID administration, the physical appearance of all groups was obviously normal and the average body weight of the groups was not significantly different. However, abnormal fat in the liver was observed only in the hepatic samples of mefenamic acid, ibuprofen and meloxicam treated mice (Fig. 1). Fig. 2 shows the effects of NSAIDs on the expression of ARA-cyp450 genes in mouse kidneys. Expression of the EET synthesizing gene cyp2c29 was induced by NSAIDs by more than 2 fold compared to the control group. Expression of cyp2j5 was reduced in the kidneys after diclofenac (1.03 fold), mefenamic acid (1.4 fold), ibuprofen (2.1 fold) and meloxicam (5.7 fold) treatment compared to the control group. Only meloxicam showed a statistically significant difference on renal cyp2j5 gene expression (p value > 0.05). In addition, NSAIDs significantly downregulated the expression of the EET metabolizing gene epdhx2 by more than 2.5 fold compared to the control group (p value < 0.05). On the other hand, NSAIDs significantly downregulated expression of 20-HETE synthesizing cyp4a12 and cyp1a2 compared to the control group (p value < 0.05), with the strongest effect seen in mefenamic acid treatment (Fig. 2). Among the tested NSAIDs, mefenamic acid and meloxicam influenced renal expression of ARA-metabolizing cyp450 genes more strongly than diclofenac and ibuprofen. Collectively, NSAIDs increased the ratio of gene expression of EETs compared to 20-HETE synthesizing enzymes in the kidney samples. In the heart, all NSAIDs upregulated cyp4a12 gene expression by more than 2.3 fold compared to the control group (Fig. 3). The highest influence on cyp4a12 gene expression was observed with mefenamic acid treatment, which upregulated cyp4a12 gene expression by more than 13 fold compared to the control group. Furthermore, mefenamic acid, ibuprofen, and meloxicam induced significant cyp1a2 gene expression compared to Bestatin hydrochloride the control group (p value <0.05), while diclofenac did not affect cyp4a12 expression. Regarding the gene expression of EET synthesizing and metabolizing genes, diclofenac upregulated cyp2j5 and cyp2c29 gene by 2.4 and 3 fold, respectively, while other NSAIDs did not alter significantly alter the expression of these genes compared to the control group (p value > 0.05). The gene expression of epdhx2 was not altered significantly in the cardiac samples after NSAID treatment (p value <0.05). Based on these results, NSAIDs increased the ratio of gene expression of 20-HETE compared to EETs synthesizing enzymes in the cardiac samples. These molecular alterations of cyp450 genes in the hearts were associated with decreased expression of the cardiotoxicity biomarker GATA4 gene in the hearts of NSAID-treated mice by more than 2.1 fold compared to the control group (Fig. 4A), and significant induction of the inflammatory biomarker cox2 was observed (ANOVA, p value <0.05, Fig. 4B). Although the expression heart failure biomarkers ANP and BNP genes was reduced after NSAID treatment, this reduction failed to reach the statistical difference (p value > 0.05, Fig. 4C).