Piper auritum ethanol extract is a potent antimutagen against food-borne aromatic amines. Mechanisms of action and chemical composition.

An ethanol extract of Piper auritum leaves (PAEE) inhibits the mutagenic effect of three food-borne aromatic amines (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) in the TA98 Salmonella typhimurium strain. Preincubation with MeIQx demonstrated in mutagenesis experiments that inhibition of Cytochrome P450 (CYP), as well as direct interaction between component(s) of the plant extract with mutagens, might account for the antimutagenic observed effect. Gas chromatography/mass spectrometry analysis revealed that safrole (50.7%), α-copaene (7.7%), caryophyllene (7.2%), ß-pinene (4.2%), γ-terpinene (4.1%) and pentadecane (4.1%) as the main components of PAEE. Piper extract and safrole were able to inhibit the rat liver microsomal CYP1A1 activity that participates in the amines metabolism, leading to the formation of the ultimate mutagenic/ molecules. According to this, safrole and PAEE inhibited MeIQx mutagenicity but not that of the direct mutagen 2-nitrofluorene. No mutagenicity of plant extract or safrole was detected. This study show that PAEE and its main component safrole are associate with the inhibition of heterocyclic amines activation due in part to the inhibition of CYP1A subfamily activity.

Neuroinflammation is able to downregulate cytochrome P450 epoxygenases 2J3 and 2C11 in the rat brain.

Cytochrome P450 (CYP) epoxygenases have been considered the main producers of epoxyeicosatrienoic acids (EETs) through the oxidation of arachidonic acid (AA). EETs display various biological properties, notably their powerful anti-inflammatory activities. In the brain, EETs have proven to be neuroprotective and to improve neuroinflammation. However, it is known that inflammation could modify CYP expression. We have previously reported that an inflammatory process in astrocytes is able to down-regulate CYP2J3 and CYP2C11 mRNA, protein levels, and activity (Navarro-Mabarak et al., 2019). In this work, we evaluated the effect of neuroinflammation in protein expression of CYP epoxygenases in the brain. Neuroinflammation was induced by the intraperitoneal administration of LPS (1 mg/kg) to male Wistar rats and was corroborated by IL-6, GFAP, and Iba-1 protein levels in the cortex over time. CYP2J3 and CYP2C11 protein levels were also evaluated in the cortex after 6, 12, 24, 48, and 72 h of LPS treatment. Our results show for the first time that neuroinflammation is able to downregulate CYP2J3 and CYP2C11 protein expression in the brain cortex.

Reduction in CYP1A1 and 2B2 activity at low oxygen tension.

The Cytochrome P450 (CYP) enzyme family comprises a wide array of monooxygenases involved in the oxidation of endobiotic and xenobiotic molecules. The active site of a CYP enzyme contains an iron protoporphyrin center coordinated to a cysteine thiolate, and then, molecular oxygen is associated with the iron to be converted into dioxygen complex plus substrate. Reduction by CYP reductase expedites hydroxylation of the compound. In this oxidation reaction, insufficient oxygen molecules would affect enzyme catalysis. Nevertheless, biochemical data about CYP kinetics at low oxygen concentrations are not available. In this work, we present the results on the variation in rat liver microsomal CYP Vmax app and Km app under normal and hypoxic conditions. Using alkoxyresorufin molecules as substrates, the Vmax/Km ratios for resorufin production decreased from 426 to 393 for CYP1A1 and from 343 to 202 for CYP2B1 at a low oxygen concentration (4.1 ppm) compared to the ratios observed at a normal oxygen concentration (6.5 ppm). Additionally, the bacterial mutagenicity of 2-aminoanthracene and cyclophosphamide, decreased by 32% and 42%, respectively, at low oxygen concentrations. These results support the hypothesis that low oxygen availability is implicated in the low efficiency of substrate oxidation by CYP.

Modulation of the rat hepatic cytochrome P4501A subfamily using biotin supplementation.

Studies have found that biotin favors glucose and lipid metabolism, and medications containing biotin have been developed. Despite the use of biotin as a pharmacological agent, few studies have addressed toxicity aspects including the possible interaction with cytochrome P450 enzyme family. This study analyzed the effects of pharmacological doses of biotin on the expression and activity of the cytochrome P4501A subfamily involved in the metabolism of xenobiotics. Wistar rats were treated daily with biotin (2 mg/kg, i.p.), while the control groups were treated with saline. All of the rats were sacrificed by cervical dislocation after 1, 3, 5, or 7 days of treatment. CYP1A1 and CYP1A2 mRNAs were modified by biotin while enzyme activity and protein concentration were not affected. The lack of an effect of biotin on CYP1A activity was confirmed using other experimental strategies, including (i) cotreatment of the animals with biotin and a known CYP1A inducer; (ii) the addition of biotin to the reaction mixtures for the measurement of CYP1A1 and CYP1A2 activities; and (iii) the use of an S9 mixture that was prepared from control and biotin-treated rats to analyze the activation of benzo[a]pyrene (BaP) into mutagenic metabolites using the Ames test. The results suggest that biotin does not influence the CYP1A-mediated metabolism of xenobiotics.

Bergamottin is a competitive inhibitor of CYP1A1 and is antimutagenic in the Ames test.

Grapefruit juice (GJ) is a well known Cytochrome P450 (CYP) inhibitor; CYP3A is one of the most affected subfamily leading to anticarcinogenic and antimutagenic effects when GJ is administered to experimental animals in combination with mutagenic/carcinogenic agents metabolized by CYP3A. Bergamottin, naringin and dihydroxybergamottin are three main constituents contained within GJ and their inhibitory effect against CYP3A4 has been well documented. Reports suggest that CYP3A is not the only one affected but CYP1A and 2B are also affected by GJ. To explore this last possibility in depth we tested the in vitro capacity of bergamottin, naringin and dihydroxybergamottin to inhibit the activity of CYP1A and 2B subfamilies and found that bergamottin showed the strongest inhibitory effect and naringin showed no inhibition at all. Therefore, we decided to biochemically characterize the inhibitory properties of bergamottin. CYP1A1 Supersome® used in this study showed a Km(app)=0.0723 µM and a Vm(app)=6.141 µU/pmol with substrate ethoxyresorufin, and the biochemical characterization of bergamottin CYP1A1 inhibitory effect revealed that it is a competitive inhibitor with a Ki=10.703 nM. We also confirmed the antimutagenicity of this compound against the mutagenic effect of 3-methylcholanthrene and benzo[a]pyrene in the Ames test.

Correlation of the genotoxic activation and kinetic properties of Salmonella enterica serovar Typhimurium nitroreductases SnrA and cnr with the redox potentials of nitroaromatic compounds and quinones.

Bacterial nitroreductases (NRs) catalyse the oxygen-insensitive reduction of several nitro-substituted compounds and quinones. SnrA and cnr NRs have been previously identified in Salmonella enterica serovar Typhimurium; they reduce several environmental nitro compounds that display mutagenic activity in the Ames test. Although some of their biochemical properties have been reported, the substrate specificity of each protein over mutagenic nitro compounds is unknown; even more, the possible relationship between their capacity to activate nitro compounds into mutagens and the redox properties of putative substrates has been poorly investigated. We have purified SnrA and cnr and investigated their capacity to activate several mutagens in the Ames test as well as their kinetic parameters K(m) and V(max). Our results show that SnrA and cnr are able to activate 2,7-dinitrofluorene with the same efficiency and a similar mutagenic potency in the YG7132 tester strain; 1-nitropyrene and 1,3-dinitropyrene were efficiently activated by cnr, whereas 1,8-dinitropyrene, 1,6-dinitropyrene and 2-nitrofluorene were scarcely activated by either NR. The mutagenic potency of nitro compounds obtained in the presence of either enzyme correlates with their redox potential reported in the literature. On the other hand, a good correlation was obtained between the catalytic efficiency (V(max)/K(m)) of the purified cnr with the redox potential of eight molecules including nitro-substituted compounds and quinones. No correlation between redox potential and catalytic efficiency by SnrA was observed, suggesting that factors other than redox potential such as the structure of the compounds are involved in the catalytic efficiency of SnrA.

Nitrocompound activation by cell-free extracts of nitroreductase-proficient Salmonella typhimurium strains.

A characterization of nitrocompounds activation by cell-free extracts (CFE) of wild-type (AB(+)), SnrA deficient (B(+)), Cnr deficient (A(+)) and SnrA/Cnr deficient (AB(-)) Salmonella typhimurium strains has been done. The Ames mutagenicity test (S. typhimurium his(+) reversion assay) was used, as well as nitroreductase (NR) activity determinations where the decrease in absorbance generated by nitrofurantoin (NFN) reduction and NADP(H) oxidation in the presence of NFN, nitrofurazone (NFZ), metronidazole (MTZ) and 4-nitroquinoline-1-oxide (4NQO) were followed. Different aromatic and heterocyclic compounds were tested for mutagenic activation: 2-nitrofluorene (2-NF); 2,7-dinitrofluorene (2,7-DNF); 1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP); 1,6-dinitropyrene (1,6-DNP); and 1,8-dinitropyrene (1,8-DNP). Differential mutagenicity was found with individual cell free extracts, being higher when the wild type or Cnr containing extract was used; nevertheless, depending on the nitrocompound, activation was found when either NR, SnrA or Cnr, were present. In addition, all nitrocompounds were more mutagenic after metabolic activation by CFE of NR proficient strains, although AB(-) extract still showed activation capacity. On the other hand, NR activity was predominantly catalyzed by wild type CFE followed by A(+), B(+) and AB(-) extracts in that order. We can conclude that results from the Ames test indicate that Cnr is the major NR, while NFN and NFZ reductions were predominantly catalyzed by SnrA. The characterization of the residual NR activity detected by the mutagenicity assay and the biochemical determinations in the AB(-) CFE needs further investigation.