N‐demethylation of phenothiazines by lipoxygenase from soybean and human term placenta in the presence of hydrogen peroxide

Abstract

Several phenothiazine derivatives have been shown to cause reproductive toxicity. The biochemical mechanisms responsible for these effects are not fully understood at present. In this study, we investigated hydrogen peroxide-dependent oxidation of six phenothiazines by purified lipoxygenase from soybean (SLO) and human term placenta (HTPLO). Chlorpromazine was employed as the prototype phenothiazine drug. Chlorpromazine was easily demethylated releasing formaldehyde when incubated at pH 7.0 and 6.5 with SLO or HTPLO, respectively, in the presence of hydrogen peroxide. The reaction was linear with respect to time, exhibited dependence on the amount of enzyme, and the concentration of chlorpromazine and hydrogen peroxide. Under the optimal assay conditions, the estimated Vmax values for chlorpromazine N-demethylation were 139 and 7.2 nmoles/min/mg of SLO and HTPLO, respectively. Collectively, the results suggest an enzymatic nature of the reaction. In the presence of gossypol and NDGA, the classical inhibitors of different lipoxygenases, the formaldehyde production was significantly decreased, as expected. Similar to SLO, the generation of chlorpromazine cation radical, an initial oxidation product with an absorption maximum at 525 nm, was also observed with HTPLO. The radical generation was detectable only under acidic conditions (pH 3.5-4.5). The formaldehyde production was also decreased by BHT and BHA, suggesting a radical nature of the SLO-mediated chlorpromazine N-demethylation. Reduced glutathione, ascorbate, and dithiothreitol suppressed the rate of SLO-dependent formaldehyde generation, presumably due to the reduction of the cation radical back to chlorpromazine in a concentration-dependent manner. Besides chlorpromazine, SLO also oxidized promazine, triflupromazine, trifluperazine, trimeprazine, and perphenazine, albeit at different rates, in the presence of hydrogen peroxide. The evidence gathered in this in vitro study suggests that phenothiazines can undergo peroxidative N-demethylation via lipoxygenase pathway. The role of this biochemical mechanism in the in vivo developmental toxicity of phenothiazines remains to be established.

Role of Biotransformation in Conceptal Toxicity of Drugs and Other Chemicals

“…The authors reported the formation of cation free radicals from phenothiazines by SLO in the presence of H 2 O 2 . Purified HTPLO also displays similar ability to generate cation radicals from phenothiazines [185]. Furthermore, CPZ and other phenothiazines are easily N-demethylated releasing formaldehyde in the incubation media containing H 2 O 2 and either SLO or HTPLO (Table 3).…”

Lipoxygenase - a versatile biocatalyst for biotransformation of endobiotics and xenobiotics

“…Besides sulfoxidation, several phenothiazines are easily demethylated, releasing formaldehyde in the incubation media containing H 2 O 2 and either SLO or HTPLO (table 1814 A. P. Kulkarni Xenobiotic metabolism by lipoxygenases 2). Among the phenothiazines tested [129], promazine was oxidized by SLO at the highest rate while triflupromazine exhibited the slowest oxidation rate. A similar structure-activity response was displayed by HTPLO for N-demethylation of phenothiazines.…”

“…CPZ +• is relatively stable and its accumulation in reaction medium can be monitored spectrophotometrically. HTPLO can also produce CPZ +• from CPZ in the presence of H 2 O 2 [129]. Recent studies indicate that besides H 2 O 2 , the SLO-and HTPLO-catalyzed CPZ +• genesis from CPZ is also efficiently supported by PUFAs [130], cumene hydroperoxide, and tertbutyl hydroperoxide [76].…”

“…SLO, soybean lipoxygenase; HTPLO, human term placental lipoxygenase; CHP, cumene hydroperoxide, TBHP, tert-butyl hydroperoxide, N.D., not determined [from refs 76,129]…”

Foreign Compounds and Intermediary Metabolism: Sulfoxidation Bridges the Divide

“…However, they too have been found to be able to undertake the metabolism of a variety of xenobiotic substrates. In various studies, lipoxygenase was shown to act upon several tricyclic drugs including amitryptiline, chlorpromazine, doxepin, imipramine, promazine and promethazine [87][88][89][90] in addition to oxyphenbutazone [91] and phenytoin [92]. Similarly, prostaglandin H-synthase catalysed the biotransformation of many xenobiotic compounds as well as the drugs, aminopyrine [93], diethylstilboestrol [94,95], oestradiol [96], phenylbutazone [97] and sulindac sulfide [98,99].…”