Retrorsine in breast milk influences copper handling in suckling rat pups

To explore the hypothesis that a second xenobiotic agent is required with excess copper to produce Indian Childhood Cirrhosis, this study investigated the effect of the pyrrolizidine alkaloid retrorsine fed to the mother during the suckling period upon the serial changes in neonatal copper status.

Female Wistar rats with new-born litters were fed either a control or a retrorsine (50 mg/kg) diet. At 0, 4, 8, 11, 15, 18 and 21 days, pups from each litter were weighed, sacrificed and their livers removed for copper, DNA and metallothionein analysis. Serum samples were assayed for caeruloplasmin oxidase activity and albumin.

1) Higher than adult level of hepatic copper in normal rats which rose post-natally before declining from day 11 after birth, 2) raised hepatic copper concentrations and total copper in the retrorsine group from day 15; levels were higher than adult at birth, 3) reduced serum caeruloplasmin oxidase activity and albumin levels in retrorsine group, but both groups lower than adult, 4) lower hepatic metallothionein levels in retrorsine group, but both groups higher than adult, and 5) reduced liver DNA in the retrorsine group when expressed as total DNA and per gram of tissue. These changes were not secondary to under-nutrition as a small study on under-nourished rat neonates showed that copper handling is not significantly altered when compared to well-nourished rats.

Retrorsine passing to rat neonates via breast milk causes: 1) the accumulation of hepatic copper, 2) impairment of the rise in serum caeruloplasmin, which could indicate a decline in synthesis or failure of copper incorporation into the apo-protein, 3) a decrease in hepatic metallothionein and serum albumin levels, again suggesting diminished protein synthesis, and 4) reduced hepatic DNA indicative of decreased cell number but increased cell size. Accumulation of liver copper but reduction of copper-binding proteins could result in free copper and explain the synergistic hepatotoxicity of copper and retrorsine.