The choroid plexus (CP) and blood-brain barrier (BBB) control the movement of several drugs and endogenous compounds between the brain and systemic circulation. The multidrug resistance associated protein (Mrp) efflux transporters form part of these barriers. Several Mrp transporters are positively regulated by the transcription factor nuclear factor erythroid-2-related factor (Nrf2) in liver. The Mrps, Nrf2 and Nrf2-dependent genes are cytoprotective and our aim was to examine basal gender differences in expression of Mrp transporters, Nrf2 and Nrf2-dependent genes (Nqo1 and Ho-1) in the brain-barriers. Previous studies have shown higher expression of Mrp1, Mrp2 and Mrp4 in female mouse liver and kidney. We hypothesized that similar renal/hepatic gender-specific patterns are present in the brain-barrier epithelia interfaces. qPCR and immunoblot analyses showed that Mrp4, Ho-1 and Nqo1 expression was higher in female CP. Mrp1, Mrp2 and Nrf2 expression in the CP had no gender pattern. Female Mrp1, Mrp2 and Mrp4 mouse brain expressions in remaining brain areas, excluding CP, were higher than male. Functional analysis of Mrp4 in CP revealed active accumulation of the Mrp4 model substrate fluo-cAMP. WT female CP had 10-fold higher accumulation in the vascular spaces than males and 60% higher than Mrp4−/− females. Probenecid blocked all transport. Methotrexate did as well except in Mrp4−/− females where it had no effect, suggesting compensatory induction of transport occurred in Mrp4−/−. Collectively, our findings indicate significant gender differences in expression of Mrp transporters and cytoprotective genes in the CP and BBB.

The choroid plexus (CP) or blood‐cerebrospinal fluid barrier (BCSFB) and blood‐brain barrier (BBB) control the movement of drugs and metabolites between the brain and systemic circulation. Gender differences in drug excretion and detoxification mechanisms can affect therapeutic and toxic responses. For example, male mice are more susceptible to acetaminophen (APAP) induced hepatotoxicity. The Abc transporters and one of their transcription factors, nuclear factor erythroid‐2‐related factor (Nrf2), play roles in the susceptibility to APAP induced hepatotoxicity. Our aim was to examine gender differences in expression of Abc transporters, Nrf2 and related stress genes (NAD(P)H quionine oxidoreductase‐1, Nqo1, and heme oxigenase‐1, Ho‐1) in the brain and CP. We hypothesized that hepatic/renal gender‐specific patterns of these cytoprotective genes would also be present in the blood brain interfaces. In female mice, as compared to male, Abcc1‐2 and Abcc4 transporters are more strongly expressed brain, supporting our hypothesis. qRT‐PCR analysis revealed higher expression of Abcc1‐2 and Nrf2 in the female brain, but no differences in the CP. Abcc4, Ho‐1 and Nqo1 showed higher expression in female in both brain interfaces. These differences may contribute to APAP potential for neurotoxicity. We examined the differences in mRNA expression of cytoprotective genes at the brain interfaces of WT and Nrf2−/− mice following a hepatotoxic dose of APAP (400 mg/kg). APAP enhanced the expression of Abcc2 and Abcc4 in brains of male WT mice, but reduced the expression of these transporters in male Nrf2−/− mice. No changes were observed in the brains of either genotype of female mice. These data suggest that the expression of these transporters in the female mouse brain are not altered by a hepatotoxic dose of APAP and that transcription factors other than Nrf2 may be responsible for the regulation of Abc transporters in the female brain.Support or Funding InformationSupported by NSF and NIH

Multidrug efflux transporters of the ATP‐Binding cassette (ABC) family, ABCC1 (Mrp1) and ABCC4 (Mrp4) located on the basolateral membrane of the CP, play important roles in clearing the brain of unwanted substances and protecting it from potentially harmful material in the circulation. Previous research has shown gender‐specific patterns and compensatory responses in liver and kidney. However, little is known about the gender differences and their function in brain. In the present study we examine Mrp1 and Mrp4 mRNA expression in CP using a Mrp4 knockout mouse (Mrp4‐/‐) model and also investigate whether Abcc transporters are expressed in a gender dependent pattern as in kidney and liver. We hypothesized that the female predominant Mrp1 and Mrp4 expression will remain in CP and that in the absence of Mrp4, the activity of Mrp1 will increase. qPCR and immunoblot analysis showed that Mrp4 mRNA and protein are expressed at much higher levels in the female than in male CP. Immunoblot analysis on CP of Mrp4‐/‐ mice showed higher protein expression of Mrp1 in males supporting our hypothesis that the activity of the Mrp1 would increase. Abcc transporters share a wide variety of substrates: therefore, compensation by Mrp1 in the absence of Mrp4 and gender divergent transporter expression could manifest in differential disposition of endogenous substrates, toxicants, and therapeutic drugs, from the CSF to the blood. These sex‐specific differences in the CP highlight the need to evaluate sex differences in neurological disorders, especially those that differ in prevalence and symptoms between men and women.

Acetaminophen (APAP) overdose can cause liver and kidney necrosis. Male mice are known to be more susceptible to APAP hepatotoxicity than females, suggesting that gender‐related genotypic traits play a role in APAP toxicity. Gender‐related differences in susceptibility to APAP nephrotoxicity in mice are also well documented and attributed to differential renal expression of CYP2E1. Recent studies in our laboratory investigated the role of the transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2), the efflux transporter multidrug resistance‐associated protein 4 (Mrp4), and the drug metabolizing enzyme flavin‐containing mono‐oxygenase 3 (Fmo3) in sex‐dependent susceptibility to APAP‐induced hepatotoxicity in mice. However, the expression and potential role of Nrf2, Fmo3, and Mrp4 in the responsiveness of male and female mouse kidneys to toxic doses of APAP has not been investigated. In the current study, we examined gene expression and protein levels of Nrf2, Mrp4, and Fmo3 in the kidneys of APAP (400 mg/kg) and vehicle treated male and female mice. Plasma alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels were measured to assess liver and renal damage. Confirmation of tissue damage was made by histopathological analysis. Immunoblots and qRT‐PCR analysis revealed that basal renal Fmo3 gene and protein expression is significantly greater in male than female mice, while Mrp4 expression is higher in females. No differences in basal Nrf2 gene and protein expression were observed among genders. High plasma ALT values and histopathological analysis indicated the presence of hepatotoxicity in both males and females receiving APAP. Renal damage was not evident, as BUN values were within the normal range for both genders and the kidney histology was normal. In response to APAP treatment, no changes in renal expression of Mrp4 or Fmo3 were observed in either gender, consistent with the absence of renal pathology. However, renal Nrf2 gene and protein expression decreased significantly in female mice after APAP dosing. In conclusion, this study documents for the first time the difference in basal renal expression levels of Nrf2 and Fmo3 in male and female mice. Further studies will investigate the expression and regulation of these genes at doses of APAP resulting in renal oxidative stress and toxicity and their potential involvement in the sex‐dependent responses to APAP induced renal damage.Support or Funding InformationSupported by NSF and NIH