Microcystin-RR is a biliary toxin selective for neonatal extrahepatic cholangiocytes

Abstract

Background & Aims

Biliary atresia is a fibrosing cholangiopathy affecting neonates that is thought to result from a prenatal environmental insult to the bile duct. Biliatresone, a plant toxin with an α-methylene ketone group, was previously implicated in biliary atresia in Australian livestock, but is found in a limited location and is unlikely to be a significant human toxin. We hypothesized that other unsaturated carbonyl compounds, some with the potential for significant human exposure, might also be biliary toxins.

Methods

We focused on the family of microcystins, cyclic peptide toxins from blue-green algae that are found worldwide, particularly during harmful algal blooms. We used primary extrahepatic cholangiocyte spheroids and extrahepatic bile duct explants from both neonatal [a total of 86 postnatal day (P) 2 mouse pups and 18 P2 rat pups (n = 8–10 per condition for both species)] and adult rodents [a total of 31 P15–18 mice (n = 10 or 11 per condition)] to study the biliary toxicity of microcystins and potential mechanisms involved.

Results

Results showed that 400 nM microcystin (MC)-RR, but not six other microcystins or the related algal toxin nodularin, caused >80% lumen closure in cell spheroids made from extrahepatic cholangiocytes isolated from 2–3-day-old mice (p <0.0001). By contrast, 400 nM MC-RR resulted in less than an average 5% lumen closure in spheroids derived from neonatal intrahepatic cholangiocytes or cells from adult mice (p = 0.4366). In addition, MC-RR caused occlusion of extrahepatic bile duct explants from 2-day-old mice (p <0.0001), but not 18-day-old mice. MC-RR also caused a 2.3-times increase in reactive oxygen species in neonatal cholangiocytes (p <0.0001), and treatment with N-acetyl cysteine partially prevented microcystin-RR-induced lumen closure (p = 0.0004), suggesting a role for redox homeostasis in its mechanism of action.

Conclusions

We identified MC-RR as a selective neonatal extrahepatic cholangiocyte toxin and suggest that it acts by increasing redox stress.

Impact and implications:

The plant toxin biliatresone causes a biliary atresia-like disease in livestock and vertebrate animal model systems. We tested the widespread blue-green algal toxin, microcystin-RR, another highly electrophilic unsaturated carbonyl compound that is released during harmful algal blooms, and found that it was also a biliary toxin with specificity for neonatal extrahepatic cholangiocytes. This work should drive further animal studies and, ultimately, studies to determine whether human exposure to microcystin-RR causes biliary atresia.