Up- and down-modulation of liver cytochrome P450 activities and associated events in two murine malaria models

Abstract

Background: The mechanisms by which malaria up and down-regulates CYP activities are not understood yet. It is also unclear whether CYP activities are modulated during non-lethal malaria infections. This study was undertaken to evaluate the time course of CYP alterations in lethal (Plasmodium berghei ANKA) and non-lethal (Plasmodium chabaudi chabaudi) murine malaria. Additionally, hypotheses on the association of CYP depression with enhanced nitric oxide (NO) production, and of CYP2a5 induction with endoplasmic reticulum dysfunction, enhanced haem metabolism and oxidative stress were examined as well.

Methods: Female DBA-2 and C57BL/6 mice were infected with P.berghei ANKA or P. chabaudi and killed at different post-infection days. Infection was monitored by parasitaemia rates and clinical signs. NO levels were measured in the serum. Activities of CYP1a (ethoxyresorufin-O-deethylase), 2b (benzyloxyresorufin-O-debenzylase), 2a5 (coumarin-7-hydroxylase) and uridine-diphosphoglucuronyl-transferase (UGT) were determined in liver microsomes. Glutathione-S-transferase (GST) activity and concentrations of gluthatione (GSH) and thiobarbituric acid-reactive substances (TBARS) were determined in the liver. Levels of glucose-regulated protein 78 (GRP78) were evaluated by immunoblotting, while mRNAs of haemoxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) were determined by quantitative RT-PCR.

Results: Plasmodium berghei depressed CYP1a and 2b and induced 2a5 in DBA-2 mice. In P.berghei-infected C57BL/6 mice CYP activities remained unaltered. In both strains, GST and UGT were not affected by P.berghei. Plasmodium c. chabaudi depressed CYP1a and 2b and induced 2a5 activities on the day of peak parasitaemia or near this day. CYP2a5 induction was associated with over-expression of HO-1 and enhanced oxidative stress, but it was not associated with GRP78 induction, a marker of endoplasmic reticulum stress. Plasmodium chabaudi increased serum NO on days near the parasitaemia peak in both strains. Although not elevating serum NO, P.berghei enhanced iNOS mRNA expression in the liver.

Conclusion: Down-regulation of CYP1a and 2b and induction of 2a5 occurred in lethal and non-lethal infections when parasitaemia rates were high. A contribution of NO for depression of CYP2b cannot be ruled out. Results were consistent with the view that CYP2a5 and HO-1 are concurrently up-regulated and suggested that CYP2a5 induction may occur in the absence of enhanced endoplasmic reticulum stress.

Figure 1

Effects of P.berghei infection on CYP activities, NO production and liver weights in DBA-2 mice. Time course of changes of liver CYP activities (CYP1a: EROD, 2b: BROD, 2a5: COH), NO serum levels, and liver and spleen weights in DBA-2 mice infected with P.berghei (ANKA). Panel A shows NO serum levels (μM) and parasitaemia rates (%PE). Results in Panel B (enzyme activities) and C (liver and spleen weights) are shown as % of mean values for non-infected controls (day 0 = 100) and comparisons were made by ANOVA and Dunnett's post-hoc test. In panels B and C statistical evaluation was made with untransformed data. Differences (P < 0.05) from non-infected control group (day 0) are indicated by an asterisk (*) superscript. Panels B and C control values (100%) are as follows (mean ± S.E.M.): EROD = 94.7 ± 41 pmol resorufin/mg protein/min; BROD = 74.6 ± 21 pmol resorufin/mg protein/min; COH = 193.9 ± 27 pmol umbelliferone/mg protein/min; liver wt = 0.99 ± 0.02 g; spleen wt = 0.08 ± 0.04 g. Control NO = 35.4 ± 2.8 μM (Panel A). Numbers of mice used (N) are as follows: PID0 = 15; PID4 = 10; PID8 = 10; PID12 = 10; PID16 = 11; PID20 = 14. Of 9 infected mice that were not killed (not included), 2 died on PID22, and 7 on PID24. No infected DBA-2 exhibited neurological signs of cerebral malaria.

Figure 2

Effects of P.berghei infection on CYP activities, NO production and liver weights in C57BL/6 mice. Time course of changes of liver CYP activities (CYP1a: EROD, 2b: BROD, 2a5: COH), NO serum levels, and liver and spleen weights in C57BL/6 mice infected with P.berghei (ANKA). Panel A shows NO serum levels (μM) and parasitaemia rates (%PE). Results in Panel B (enzyme activities) and C (liver and spleen weights) are shown as % of mean values for non-infected controls (day 0 = 100) and comparisons were made by ANOVA and Dunnett's post-hoc test. In panels B and C statistical evaluation was made with untransformed data. Differences (P < 0.05) from non-infected control group (day 0) are indicated by an asterisk (*) superscript. Panels B and C control values (100%) are as follows (mean ± S.E.M.): EROD = 103.1 ± 33.5 pmol resorufin/mg protein/min; BROD = 87.3 ± 12.3 pmol resorufin/mg protein/min; COH = 9.81 ± 2.5 pmol umbelliferone/mg protein/min; liver wt = 0.99 ± 0.05 g; spleen wt = 0.09 ± 0.01 g. Control NO = 37.95 ± 2.4 μM (Panel A). Numbers of mice used (N) are as follows: PID0 = 5; PID2 = 7; PID4 = 7; PID6 = 7; PID8 = 7; PID10 = 3. Numbers of infected mice which died (not included): PIDs8/9 = 5, PIDs9/10 = 13. From day 8 onwards, all infected mice that were killed or died presented neurological signs of CM.

Figure 3

Time course of non-lethal malaria (P.chabaudi chabaudi) infection in female DBA-2 and C57BL/6 mice. Parasitaemia rates (%PE) were determined following inoculation of P. c. chabaudi (2 × 10⁷ PE i.p.) on infection day 0 (PID0). Bar heights are means ± S.E.M. of % of parasitized erythrocytes (%PE). Number of mice used (N): DBA-2 = 5; C57BL/6 = 5.

Figure 4

Effects of P. c. chabaudi infection on CYP activities, NO production and liver weights in DBA-2 mice. Time course of changes of liver CYP activities (CYP1a: EROD, 2b: BROD, 2a5: COH), NO serum levels, and liver and spleen weights in DBA-2 mice infected with P.chabaudi chabaudi. Panel A shows NO serum levels (μM) and parasitaemia rates (%PE). Results in panels B (enzyme activities) and C (liver and spleen weights) are shown as % of mean values for non-infected controls (day 0 = 100) and comparisons were made by ANOVA and Dunnett's post-hoc test. In panels B and C statistical evaluation was made with untransformed data. Differences (P < 0.05) from non-infected control group (day 0) are indicated by an asterisk (*) superscript. Panels B and C control values (100%) are as follows (mean ± S.E.M.): EROD = 130.8 ± 6.7 pmol resorufin/mg protein/min; BROD = 73.7 ± 3.1 pmol resorufin/mg protein/min; COH = 149.4 ± 8.0 pmol umbelliferone/mg protein/min; liver wt = 1.08 ± 0.01 g; spleen wt = 0.11 ± 0.005 g. Control NO = 42.25 ± 1.7 μM (Panel A). Numbers of mice used (N) are as follows: PID0 = 8; PID2 = 8; PID5 = 8; PID6 = 8; PID10 = 8, PID14 = 8; PID19 = 8.

Figure 5

Effects of P. c. chabaudi infection on CYP activities, NO production and liver weights in C57BL/6 mice. Time course of changes of liver CYP activities (CYP1a: EROD, 2b: BROD, 2a5: COH), NO serum levels, and liver and spleen weights in C57BL/6 mice infected with P. chabaudi chabaudi. Panel A shows NO serum levels (μM) and parasitaemia rates (%PE). Results in Panel B (enzyme activities) and C (liver and spleen weights) are shown as % of mean values for non-infected controls (day 0 = 100) and comparisons were made by ANOVA and Dunnett's post-hoc test. In panels B and C statistical evaluation was made with untransformed data. Differences (P < 0.05) from non-infected control group (day 0) are indicated by an asterisk (*) superscript. Panels B and C control values (100%) are as follows (mean ± S.E.M.): EROD = 182.4 ± 5.8 pmol resorufin/mg protein/min; BROD = 121.2 ± 6.0 pmol resorufin/mg protein/min; COH = 16.5 ± 1.2 pmol umbelliferone/mg protein/min; liver wt = 0.84 ± 0.02 g; spleen wt = 0.07 ± 0.003 g. Control NO = 51.12 ± 2.36 μM (Panel A). Numbers of mice used (N) are as follows: PID0 = 10; PID2 = 14; PID5 = 10; PID6 = 6, PID10 = 6; PID14 = 6; PID19 = 8.

Figure 6

Effects of P. berghei infection on iNOS mRNA expression in mice liver and spleen. Expression of iNOS mRNA in the liver and spleen of DBA-2 (A and B, N = 3) and C57BL/6 (C and D, N = 4) mice infected with P. berghei (ANKA), when parasitaemia rates rose to levels >30%PE (DBA-2) on PID20 or >20%PE (C57BL/6) on PID10 and respective non-infected controls (N = 3). For comparative purposes, levels of iNOS mRNA were also determined in the liver (A, N = 6) and spleen (B, N = 5) of non-infected DBA-2 mice treated with E. coli LPS (5 mg/kg i.p. 12 hours earlier). Column height represents mean ± S.E.M. for relative levels of mRNA. Differences (Mann-Whitney U test, P < 0.05) between infected (malaria) and non-infected control (Ctrl) are indicated by an asterisk (*). Quantification of mRNA was by real time RT-PCR.

Figure 7

Effects of P. berghei infection on haemoxygenase-1 mRNA expression in the liver of DBA-2 mice. Expression of HO-1 mRNA in the liver of DBA-2 mice infected with P.berghei (ANKA), when parasitaemia rates rose to levels >30%PE on PID20, and respective non-infected controls. Column height represents mean ± S.E.M. for relative levels of mRNA of 6 mice. Differences (Mann-Whitney U test, P < 0.05) between infected (malaria) and non-infected controls (Ctrl) are indicated by an asterisk (*). Quantification of mRNA was by real time RT-PCR.

Figure 8

Effect of P. berghei infection on GRP78 protein expression in the liver of DBA-2 mice. Immunoblot analysis - with a polyclonal antibody against GRP78 - of liver microsomes from female DBA-2 mice infected with P. berghei (Malaria) on PID20, non-infected controls (Control) and non-infected mice treated with pyrazole (Pyrazole), 100 mg/kg body wt i.p. Densitometric analysis is shown in the lower panel. Height of histogram bars are means ± S.D. of arbitrary optical density units. Difference from non-infected control group is indicated by an asterisk (*, P < 0.05, Mann-Whitney U test). 50 μg of microsomal protein from a different mouse was applied on each lane. The immonoblot shown in the figure is representative of others with similar results.