Identification of Enzymes Involved in the Metabolism of Atrazine, Terbuthylazine, Ametryne, and Terbutryne in Human Liver Microsomes

Abstract

Compounds of the s-triazine family are among the most heavily used herbicides over the last 30 years. Some of these derivatives are suspected to be carcinogens. In this study the identity of specific phase-I enzymes involved in the metabolism of s-triazine derivatives (atrazine, terbuthylazine, ametryne, and terbutryne) by human liver microsomes was determined. Kinetic studies demonstrated biphasic kinetics for all pathways examined (S-oxidation, N-dealkylation, and side-chain C-oxidation). Low K(m) values were in a range of about 1-20 microM, whereas high K(m) values were up to 2 orders of magnitude higher. For a correlation study, 30 human liver microsomal preparations were screened for seven specific P450 activities, and these were compared to activities for the metabolites derived from these s-triazines. A highly significant correlation in the high-affinity concentration range was seen with cytochrome P450 1A2 activities. Chemical inhibition was most effective with alpha-naphthoflavone and furafylline at low s-triazine concentrations and additionally with ketoconazole and gestodene at high substrate concentrations. Studies with 10 heterologously expressed P450 forms demonstrated that several P450 enzymes are capable of oxidizing these s-triazines, with different affinities and regioselectivities. P450 1A2 was confirmed to be the low-K(m) P450 enzyme involved in the metabolism of these s-triazines. A potential participation of flavin-containing monooxygenases (FMOs) in sulfoxidation reactions of the thiomethyl derivatives ametryne and terbutryne in human liver was also evaluated. Sulfoxide formation in human liver microsomes as a function of pH, heat, and chemical inhibition indicated no significant involvement of FMOs. Finally, purified recombinant FMO3, the major FMO in human liver, exhibited no significant activity (< 0.1 nmol (nmol of FMO3)-1 min-1) in the formation of the parent sulfoxides of ametryne and terbutryne. Therefore, P450 1A2 alone is likely to be responsible for the hepatic oxidative phase-I metabolism of the s-triazine derivatives in exposed humans.

International regulatory needs for development ofan IATA for non-genotoxic carcinogenic chemical substances

“…Induction of CYP 2B10 associated with CAR activation (Ren et al, 2010) In vitro liver microsomal CYP 1A2 induction (Lang et al, 1996(Lang et al, , 1997 Assessment of effects on CYP 1A1 and CYP 2B (Rahden-Staron et al, 2001) Level 0 (Q)SAR and structure activity information (Benigni, 2012;Serafimova et al, 2007; SAR and grouping based upon mammary gland tumor induction by various striazine compounds in rats (US EPA, 2002) Existing literature data (Rakitsky et al, 2000;NTP, 2008NTP, , 2011IARC, 1991 is important, also low probability of false negatives is critical.…”

Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?

“…CYP1A2 has a major role in the metabolism of many important chemicals such as caffeine (Butler et al, 1989;Tassaneeyakul et al, 1992), phenacetin (Sesardic et al, 1990;Venkatakrishnan et al, 1998), theophylline (Sarkar & Jackson, 1994;Tjia et al, 1996), clozapine (Fang et al, 1998), melatonin (Facciolá et al, 2001;von Bahr et al, 2000), and tizanidine (Granfors et al, 2004a;Granfors et al, 2004b). CYP1A1 is a major enzyme in the metabolism of a number of insecticides and herbicides (Lang et al, 1997;Tang et al, 2002;Abass et al, 2010;Abass et al, 2007c). CYP1A2 mediates herbicides (Lang et al, 1997;Abass et al, 2007c;Nagahori et al, 2000), insecticides (Tang et al, 2002;Stresser & Kupfer, 1998;Foxenberg et al, 2007;Mutch & Williams, 2006), and pyrethroids metabolism (Scollon et al, 2009).…”

“…CYP1A1 is a major enzyme in the metabolism of a number of insecticides and herbicides (Lang et al, 1997;Tang et al, 2002;Abass et al, 2010;Abass et al, 2007c). CYP1A2 mediates herbicides (Lang et al, 1997;Abass et al, 2007c;Nagahori et al, 2000), insecticides (Tang et al, 2002;Stresser & Kupfer, 1998;Foxenberg et al, 2007;Mutch & Williams, 2006), and pyrethroids metabolism (Scollon et al, 2009). …”

The effect of acute exposure to herbicide Gardoprim Plus Gold 500 SC on haematological and biochemical indicators and histopathological changes in common carp (Cyprinus carpio L.)

“…Water solubility of terbuthylazine is 8.5 mg·l -1 at 20 °C (Gangolli 1999b). There are studies on terbuthylazine toxicity to terrestrial animals (Lang et al 1997), but less is known about its toxicity to aquatic organisms, including fish (Dezfuli et al 2006).Chloracetanilide herbicides are used to control annual grasses and broad leaf weeds. Chloracetanilide metolachlor has been used to control annual grass and broad-leaved weeds following pre-emergence application for more than 20 years.…”

“…Water solubility of terbuthylazine is 8.5 mg·l -1 at 20 °C (Gangolli 1999b). There are studies on terbuthylazine toxicity to terrestrial animals (Lang et al 1997), but less is known about its toxicity to aquatic organisms, including fish (Dezfuli et al 2006).…”