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Strain- and sex-dependent circadian changes in abcc2 transporter expression: implications for irinotecan chronotolerance in mouse ileum.

Okyar A, Piccolo E, Ahowesso C, Filipski E, Hossard V, Guettier C, La Sorda R, Tinari N, Iacobelli S, Lévi F - PLoS ONE (2011)

Bottom Line: Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF(1) (p = 0.0006).Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan.This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U776 Rythmes Biologiques et Cancers, Hôpital Paul Brousse, Villejuif, France.

ABSTRACT

Background: ATP-binding cassette transporter abcc2 is involved in the cellular efflux of irinotecan. The drug is toxic for mouse ileum, where abcc2 is highly expressed. Here, we investigate whether circadian changes in local abcc2 expression participate in the circadian rhythm of irinotecan toxicity for ileum mucosa, and further assess whether genetic background or sex modify this relation.

Methodology/principal findings: Ileum mucosa was obtained every 3-4 h for 24 h in male and female B6D2F(1) and B6CBAF(1) mice synchronized with light from Zeitgeber Time (ZT)0 to ZT12 alternating with 12 h of darkness. Irinotecan (50 mg/kg i.v. daily for 4 days) was administered at the sex- and strain-specific times corresponding to least (ZT11-15) or largest drug-induced body weight loss (ZT23-03-07). Abcc2 expression was determined with qRT-PCR for mRNA and with immunohistochemistry and confocal microscopy for protein. Histopathologic lesions were graded in ileum tissues obtained 2, 4 or 6 days after treatment. Two- to six-fold circadian changes were demonstrated for mRNA and protein mean expressions of abcc2 in mouse ileum (p<0.05). ZT12 corresponded to high mRNA and protein expressions, with circadian waveforms differing according to genetic background and sex. The proportion of mice spared from ileum lesions varied three-fold according to irinotecan timing, with best tolerability at ZT11-15 (p = 0.00003). Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF(1) (p = 0.0006).

Conclusions/significance: Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan. This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.

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Confocal immunohistochemistry imaging of abcc2 protein expression in mouse ileum, according to circadian time, genetic background and sex.A) Selected examples in male (upper row) and female (lower row) B6D2F1. B) Id in B6CBAF1. C) Histogram depicting the changes in mean (±SEM) protein expression at four ZTs associated with low or high mRNA expressions in male and female B6D2F1. D) Id in B6CBAF1. NOTE: Three-way ANOVA confirmed statistically significant differences as a function of ZT (p<0.001), strain (p = 0.048). Statistically significant interactions were detected between ZT*strain (p = 0.001), ZT*sex (p = 0.035) and ZT*strain*sex (p = 0.004).
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pone-0020393-g004: Confocal immunohistochemistry imaging of abcc2 protein expression in mouse ileum, according to circadian time, genetic background and sex.A) Selected examples in male (upper row) and female (lower row) B6D2F1. B) Id in B6CBAF1. C) Histogram depicting the changes in mean (±SEM) protein expression at four ZTs associated with low or high mRNA expressions in male and female B6D2F1. D) Id in B6CBAF1. NOTE: Three-way ANOVA confirmed statistically significant differences as a function of ZT (p<0.001), strain (p = 0.048). Statistically significant interactions were detected between ZT*strain (p = 0.001), ZT*sex (p = 0.035) and ZT*strain*sex (p = 0.004).

Mentions: Abcc2 protein was mostly expressed in the plasma membrane and occasionally in the nuclear membrane of the cells in ileum mucosa, as showed by abcc2 co-localization with a known plasma membrane marker, likeβcatenin (Figure 3A). The spatial distribution patterns were similar whatever the sex or the strain (Figures 4A and 4B). Protein expression was barely detectable in the colon, so that no quantification was attempted. The expression of abcc2 protein displayed consistent 24 h changes in male and female B6D2F1, as well as in male and female B6CBAF1, with apparent strain- and/or sex-dependent differences. Protein expression peaked at ZT12 both in male and female B6D2F1 while it was lowest at ZT15 in males and at ZT15, ZT0 and ZT3 in females (Figure 4C). The extent of circadian variation was ∼three-fold in males and ∼two-fold in females. In B6CBAF1, abcc2 protein expression was highest from ZT12 to ZT15 in males and at ZT12 in females. Lowest values were found at ZT0 in males and at ZT15 in females (Figure 4D). The range of change was ∼two fold in female mice and much weaker in males. Three-way ANOVA validated statistically significant differences as a function of ZT (p<0.001) and strain (p = 0.048), but not sex (p = 0.07). Furthermore, statistically significant interactions were found between ZT and strain (p = 0.001), and between ZT and sex (p = 0.035) as well as all three factors (p = 0.004. Overall, mean protein expression was correlated with mean mRNA expression in each group at each ZT (r = 0.52; p<0.05).


Strain- and sex-dependent circadian changes in abcc2 transporter expression: implications for irinotecan chronotolerance in mouse ileum.

Okyar A, Piccolo E, Ahowesso C, Filipski E, Hossard V, Guettier C, La Sorda R, Tinari N, Iacobelli S, Lévi F - PLoS ONE (2011)

Confocal immunohistochemistry imaging of abcc2 protein expression in mouse ileum, according to circadian time, genetic background and sex.A) Selected examples in male (upper row) and female (lower row) B6D2F1. B) Id in B6CBAF1. C) Histogram depicting the changes in mean (±SEM) protein expression at four ZTs associated with low or high mRNA expressions in male and female B6D2F1. D) Id in B6CBAF1. NOTE: Three-way ANOVA confirmed statistically significant differences as a function of ZT (p<0.001), strain (p = 0.048). Statistically significant interactions were detected between ZT*strain (p = 0.001), ZT*sex (p = 0.035) and ZT*strain*sex (p = 0.004).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108586&req=5

pone-0020393-g004: Confocal immunohistochemistry imaging of abcc2 protein expression in mouse ileum, according to circadian time, genetic background and sex.A) Selected examples in male (upper row) and female (lower row) B6D2F1. B) Id in B6CBAF1. C) Histogram depicting the changes in mean (±SEM) protein expression at four ZTs associated with low or high mRNA expressions in male and female B6D2F1. D) Id in B6CBAF1. NOTE: Three-way ANOVA confirmed statistically significant differences as a function of ZT (p<0.001), strain (p = 0.048). Statistically significant interactions were detected between ZT*strain (p = 0.001), ZT*sex (p = 0.035) and ZT*strain*sex (p = 0.004).
Mentions: Abcc2 protein was mostly expressed in the plasma membrane and occasionally in the nuclear membrane of the cells in ileum mucosa, as showed by abcc2 co-localization with a known plasma membrane marker, likeβcatenin (Figure 3A). The spatial distribution patterns were similar whatever the sex or the strain (Figures 4A and 4B). Protein expression was barely detectable in the colon, so that no quantification was attempted. The expression of abcc2 protein displayed consistent 24 h changes in male and female B6D2F1, as well as in male and female B6CBAF1, with apparent strain- and/or sex-dependent differences. Protein expression peaked at ZT12 both in male and female B6D2F1 while it was lowest at ZT15 in males and at ZT15, ZT0 and ZT3 in females (Figure 4C). The extent of circadian variation was ∼three-fold in males and ∼two-fold in females. In B6CBAF1, abcc2 protein expression was highest from ZT12 to ZT15 in males and at ZT12 in females. Lowest values were found at ZT0 in males and at ZT15 in females (Figure 4D). The range of change was ∼two fold in female mice and much weaker in males. Three-way ANOVA validated statistically significant differences as a function of ZT (p<0.001) and strain (p = 0.048), but not sex (p = 0.07). Furthermore, statistically significant interactions were found between ZT and strain (p = 0.001), and between ZT and sex (p = 0.035) as well as all three factors (p = 0.004. Overall, mean protein expression was correlated with mean mRNA expression in each group at each ZT (r = 0.52; p<0.05).

Bottom Line: Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF(1) (p = 0.0006).Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan.This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U776 Rythmes Biologiques et Cancers, Hôpital Paul Brousse, Villejuif, France.

ABSTRACT

Background: ATP-binding cassette transporter abcc2 is involved in the cellular efflux of irinotecan. The drug is toxic for mouse ileum, where abcc2 is highly expressed. Here, we investigate whether circadian changes in local abcc2 expression participate in the circadian rhythm of irinotecan toxicity for ileum mucosa, and further assess whether genetic background or sex modify this relation.

Methodology/principal findings: Ileum mucosa was obtained every 3-4 h for 24 h in male and female B6D2F(1) and B6CBAF(1) mice synchronized with light from Zeitgeber Time (ZT)0 to ZT12 alternating with 12 h of darkness. Irinotecan (50 mg/kg i.v. daily for 4 days) was administered at the sex- and strain-specific times corresponding to least (ZT11-15) or largest drug-induced body weight loss (ZT23-03-07). Abcc2 expression was determined with qRT-PCR for mRNA and with immunohistochemistry and confocal microscopy for protein. Histopathologic lesions were graded in ileum tissues obtained 2, 4 or 6 days after treatment. Two- to six-fold circadian changes were demonstrated for mRNA and protein mean expressions of abcc2 in mouse ileum (p<0.05). ZT12 corresponded to high mRNA and protein expressions, with circadian waveforms differing according to genetic background and sex. The proportion of mice spared from ileum lesions varied three-fold according to irinotecan timing, with best tolerability at ZT11-15 (p = 0.00003). Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF(1) (p = 0.0006).

Conclusions/significance: Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan. This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.

Show MeSH
Related in: MedlinePlus