The human Apolipoprotein E4 (ApoE4) variant is the strongest known genetic risk factor for Alzheimer’s disease (AD).  Cadmium (Cd) has been shown to impair learning and memory at a greater extent in humanized ApoE4 knock-in (ApoE4-KI) mice as compared to the ApoE3 (common allele)-KI mice. In this study, we determined the extent that cadmium interacts with the ApoE4 gene variants to modify the gut-liver axis, which is important for xenobiotic biotransformation and nutrient homeostasis. Large intestinal content bacterial 16S rDNA sequencing, serum lipid metabolomics, and hepatic transcriptomics were analyzed in ApoE3- and ApoE4-KI mice orally exposed to vehicle, a low dose, or a high dose of Cd in drinking water.  Aligning with the previous report showing that the ApoE4-KI males are more susceptible to cadmium-induced memory deficit, ApoE4-KI males had the most prominent changes in gut microbiota, including an up-regulation of A. muciniciphila, which is a biomarker for AD in humans, as well as predicted down-regulation of many essential microbial pathways involved in nutrient and energy homeostasis.  Serum lactate was lower only in ApoE4-KI males following Cd exposure. In the host liver, cadmium-exposed ApoE4-KI males had the most differentially regulated pathways; specifically there was an enrichment in several pathways involved in platelet activation, which is known to amplify liver damage and inflammation. These pathways were associated with up-regulated Prevotella and A. muciniphila in intestine at the Cd low dose.  Cadmium-exposed ApoE4-KI mice also had the most differentially regulated hepatic drug processing genes, and in particular, the up-regulation of Cyp2 family, Ugts, Gsts, and Slco1b2 in liver associated with the up-regulation of Clostridiaceae in the intestine at the Cd high dose.  In conclusion, Cd exposure profoundly modified the gut-liver axis in the most susceptible mouse strain to neurological damage, (ApoE4-KI males) evidenced by up-regulation in microbial AD biomarkers, reduction in energy supply-related pathways in gut and blood, and up-regulation in hepatic pathways involved in inflammation and xenobiotic biotransformation.

Total DNA was isolated from the large intestinal content of male and female ApoE3-KI and ApoE4-KI mice (n=4-5).  Briefly, samples were prepared using an E.Z.N.A. DNA Stool Kit (Omega Bio-tek Inc., Norcross, GA) per the manufacturer’s instructions. The V4 hypervariable region of 16S rDNA was amplified and sequenced using a HiSeq 2500 second generation sequencing platform (250-bp paired-end) (Novogene Corporation Inc., Sacramento, CA).

Total RNA was isolated from livers of Cd or vehicle exposed ApoE3-KI and ApoE4-KI mice (n=4 per exposure per sex) using RNA zol Bee reagent (Tel-Test Inc., Friendswood, TX). cDNA libraries were prepared using a Clontech cDNA library prep kit (Clontech Laboratories Inc., Mountain View CA), and were sequenced using a NextSeq 500 sequencing platform (75 bp paired end).