Modulation of Cytochrome P450 2A5 Activity by Lipopolysaccharide: Low-Dose Effects and Non-Monotonic Dose-Response Relationship

Submitted by sandra infurna ([email protected]) on 2016-04-18T14:25:37Z No. of bitstreams: 1 paulo_totino_etal_IOC-2015.pdf: 779725 bytes, checksum: 9034bf229a28ea9c75c5b2b112abb346 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-18T14:46:01Z (GMT) No. of bitstreams: 1 paulo_totino_etal_IOC-2015.pdf: 779725 bytes, checksum: 9034bf229a28ea9c75c5b2b112abb346 (MD5)Made available in DSpace on 2016-04-18T14:46:01Z (GMT). No. of bitstreams: 1 paulo_totino_etal_IOC-2015.pdf: 779725 bytes, checksum: 9034bf229a28ea9c75c5b2b112abb346 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Departamento de Ciências Biológicas. Laboratório de Toxicologia Ambiental. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Departamento de Ciências Biológicas. Laboratório de Toxicologia Ambiental. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Departamento de Ciências Biológicas. Laboratório de Toxicologia Ambiental. Rio de Janeiro, RJ, Brasil.Mouse cytochrome P450 (CYP) 2A5 is induced by inflammatory conditions and infectious diseases that down-regulate the expression and activity of most other CYP isoforms. Enhanced oxidative stress and nuclear factor (erythroid 2-related factor) 2 (Nrf2) transcription factor activation have been hypothesised to mediate up-regulation of CYP2A5 expression in the murine liver. The unique and complex regulation of CYP2A5, however, is far from being thoroughly elucidated. Sepsis and high doses of bacterial lipopolysaccharide (LPS) elicit oxidative stress in the liver, but depression, not induction, of CYP2A5 has been observed in studies of mice treated with LPS. The foregoing facts prompted us to evaluate the response of CYP2A5 liver activity in female DBA-2 mice over a broad range of LPS doses (0, 0.025, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 mg/kg). Cytokine levels (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon gamma, tumour necrosis factor alpha) and nitric oxide (NO) were measured in the blood serum. Activities of CYP1A (EROD) and CYP2B (BROD) in the liver were also determined for comparative purposes. LPS depressed CYP2A5 at low doses (0.025–2.0 mg/kg) but not at doses (>2 mg/kg) that increased pro-inflammatory cytokines and NO serum levels, and depressed CYP1A and CYP2B activities. Blockade of proinflammatory cytokines and the overproduction of NO induced by co-treatment with pentoxifylline and LPS and iNOS inhibition with aminoguanidine both extended down-regulation of CYP2A5 to the high dose range while not affecting LPS-induced depression of CYP1A and CYP2B. Overall, the results suggested that NO plays a role in the reversal of the low-dose LPS-induced depression of CYP2A5 observed when mice were challenged with higher doses of LPS

Activities (pmol/[min·mg protein]) of CYP2A5 (COH) and CYP1A and CYP2B (EROD and BROD, respectively) in the liver of mice treated with PTX and LPS.

<p>Monooxygenase activities were measured in the liver of female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0, 0.05, 0.1, 0.5, 1, 5, 10 or 20 mg/kg, i.p., N = 10 per dose group) alone (●) or with PTX plus LPS (2 × 100 mg/kg i.p., 60 min apart, N = 8 per LPS dose group) (▲). Data are shown as means ± SEM: <b>a</b> indicates that the mean differs from control value in the LPS-treated group; <b>b</b> indicates that the mean differs from control value in the PTX+LPS-treated group; and the asterisk (<b>*</b>) indicates that the mean value in the PTX+LPS-treated group differs from the mean value in the LPS-treated group for the same dose (ANOVA and Dunnett’s multiple comparison test followed by the Student’s <i>t</i>-test, P < 0.05).</p

Levels of pro-inflammatory cytokines in the blood serum of mice treated with pentoxifylline (PTX) and LPS.

<p>Concentrations of tumour necrosis factor alpha (TNF-α), interferon gamma (IFN- γ), interleukin (IL)-6, and IL-17A were measured in the serum of female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0, 0.05, 0.1, 0.5, 1, 5, 10 or 20 mg/kg i.p., N = 8 per dose group (except the 0 and 20 mg/kg LPS groups, N = 10) alone (●) or PTX plus LPS (2 × 100 mg/kg i.p., 60 min apart, N = 8 per dose group) (▲). Data represent means ± SEM: <b>a</b> indicates that the mean differs from the control value in the LPS-treated group; <b>b</b> indicates that the mean differs from the control value in the PTX+LPS-treated group; and the asterisk (<b>*</b>) indicates that the mean value in the PTX+LPS-treated group differs from that in the LPS-treated group for the same dose (Kruskal-Wallis and Mann-Whitney tests, P < 0.05).</p

Changes in nitric oxide (NO) concentration in the blood serum and cytochrome P450 (CYP) 2A5, CYP1A, and CYP2B activities in the liver with time elapsed after lipopolysaccharide (LPS) injection.

<p>Nitrite concentration (μM), coumarin 7-hydroxylase (COH, CYP2A5), benzyloxy-resorufin-<i>O</i>-debenzylase (BROD, CYP2B), and ethoxy-resorufin-<i>O</i>-deethylase (EROD, CYP1A) activities (pmol/[min·mg protein]) in the hepatic microsomal fraction of liver samples from female DBA-2 mice injected intraperitoneally (i.p.) with phosphate-buffered saline (control) or LPS (2, 5 or 20 mg/kg) were determined 6, 12 or 24 h after treatment. N = 7 for all groups, except for the control (0 h; N = 20) and LPS (5 mg/kg) groups at 6 h (N = 15). Data represent means ± standard error of the mean (SEM). An asterisk (<b>*</b>) indicates that the mean value differs (analysis of variance [ANOVA] and Dunnett’s multiple comparison test, P < 0.05) from that of the control group.</p

Effect of inducible NO synthase (iNOS) inhibition with aminoguanidine (AG) on LPS-induced changes in CYP2A5 (COH) and CYP1A and CYP2B (EROD and BROD, respectively) activities (pmol/[min·mg protein]) in the mouse liver.

<p>NO levels were determined in the blood serum and monooxygenase was measured in liver microsomes from female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0 or 10 mg/kg i.p.) alone or LPS (0 or 10 mg/kg i.p.) plus AG (0, 50 or 100 mg/kg i.p.). The mice per group numbered as follows: 0 (PBS alone), N = 7; LPS alone, N = 9; a50 (AG, 50 mg/kg plus PBS), N = 6; a100 (AG, 100 mg/kg plus PBS), N = 6; a50L (AG, 50 mg/kg plus LPS), N = 6; and a100L (AG, 100 mg/kg plus LPS), N = 6. Data are shown as means ± SEM. <b>a</b> and <b>b</b> above the bars indicate that the mean values differ (ANOVA and Bonferroni’s multiple comparison test, P < 0.05) from that of the group treated with PBS alone (a), LPS alone (b), or both.</p

Non-monotonic dose response of CYP2A5 activity in mouse livers after treatment with LPS.

<p>NO concentration (μM) and COH activity (pmol/[min·mg protein]) in the hepatic microsomal fraction of liver samples from female DBA-2 mice 24 h after treatment. Mice per LPS dose (mg/kg i.p.) group numbered as follows: 0 (PBS), N = 26; 0.025, N = 3; 0.05, N = 8; 0.1, N = 6; 0.2, N = 6; 0.5, N = 8; 1, N = 6; 2, N = 7; 5, N = 7; 10, N = 10; and 20, N = 7. Data represent means ± SEM. An asterisk (<b>*</b>) indicates that the mean value differs (ANOVA and Dunnett’s multiple comparison test, P < 0.05) from that of the control group.</p

Concentrations of IL-2, IL-4, and IL-10 in the blood serum of mice treated with PTX and LPS.

<p>Concentrations of cytokines were measured in the serum of female DBA-2 mice 24 h after treatment. Animals were treated with LPS (0, 0.05, 0.1, 0.5, 1, 5, 10 or 20 mg/kg i.p., N = 8 per dose group [except the 0 and 20 mg/kg LPS groups, N = 10]) alone (●) or with PTX plus LPS (2 × 100 mg/kg i.p., 60 min apart, N = 8 per LPS dose group) (▲). Data are shown as means ± SEM: <b>a</b> indicates that the mean differs from control value in the LPS-treated group; <b>b</b> indicates that the mean differs from control value in the PTX+LPS-treated group; and the asterisk (<b>*</b>) indicates that the mean value in the PTX+LPS-treated group differs from the mean value in the LPS-treated group for the same dose (Kruskal-Wallis and Mann-Whitney tests, P < 0.05).</p