Using a semi-targeted approach, we have investigated the effect of acetaminophen on circulating bile acid profiles in rats, including many known bile acids and potential isomeric structures, as well as glucuronide and sulfate conjugates. The chromatographic separation was based on an optimized reverse-phase method exhibiting excellent resolution for a complex mix of bile acids using a solid-core C18 column, coupled to a high-resolution quadrupole time-of-flight system. The semi-targeted workflow consisted of first assigning all peaks detectable in samples from 46 known bile acids contained in a standard mix, as well as additional peaks for other bile acid isomers. The presence of glucuronide and sulfate conjugates was also examined based on their elemental formulae and detectable peaks with matching exact masses were added to the list of features for statistical analysis. In this study, rats were administered acetaminophen at four different doses, from 75 to 600 mg/kg, with the highest dose being a good model of drug-induced liver injury. Statistically significant changes were found by comparing bile acid profiles between dosing levels. Some tentatively assigned conjugates were further elucidated using in vitro metabolism incubations with rat liver fractions and standard bile acids. Overall, 13 identified bile acids, 23 tentatively assigned bile acid isomers, and 9 sulfate conjugates were found to increase significantly at the highest acetaminophen dose, and thus could be linked to drug-induced liver injury.

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium represents an effective protective barrier against Cd toxicity, but it is also a target tissue that may accumulate and trap high levels of the ingested metal. Using human enterocytic‐like Caco‐2 cells, we have previously shown that Cd may induce a concentration and time‐dependent increase in 3‐(4,5‐dimethyl‐2‐thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay (MTT)‐reducing activity in differentiated cultures with correlation to ERK1/2 activation. The present study shows that (a) Zn prevents the Cd‐induced hormesis effect on MTT reduction in a concentration‐dependent manner, without inhibiting Cd‐induced ERK1/2 activation; (b) Zn also induces similar hormetic stimulation of MTT‐reducing activity but without ERK1/2 activation. The effect of both metals was sensitive to inhibitors of translation during protein synthesis. There is evidence for the involvement of reactive oxygen species (ROS) in Cd‐induced ERK1/2 activation. In contrast, the Zn effect on the MTT‐reducing activity would not be triggered by ROS but it would be sensitive to the redox state of the cell. Steps downstream ERK1/2 activation by Cd does not involve eIF4E which is rather downregulated by Cd. In conclusion, Cd and Zn both can modify translation processes during protein synthesis via different signaling cascades with crosstalk, and cross‐inhibition may occur. This phenomenon is observed over a small range of metal concentrations and is characterized by a hormesis‐like response. Considering that the hormetic effect on dehydrogenase activity could reflect an adaptive response to the metals whether cross‐inhibition is beneficial is an open question.

Cadmium is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium has the capacity to accumulate high levels of this metal. We have previously shown that Cd induces ERK1/2 activation in differentiated but not proliferative human enterocytic-like Caco-2 cells. As autophagy is a dynamic process that plays a critical role in intestinal mucosa, we aimed the present study 1) to investigate the role of p-ERK1/2 in constitutive autophagy in proliferative Caco-2 cells and 2) to investigate whether Cd-induced activation of ERK1/2 modifies autophagic activity in postconfluent Caco-2 cell monolayers. Western blot analyses of ERK1/2 and autophagic markers (LC3, SQSTM1), and cellular staining with acridine orange showed that ERK1/2 and autophagic activities both decreased with time in culture. GFP-LC3 fluorescence was also associated with proliferative cells and the presence of a constitutive ERK1/2-dependent autophagic flux was demonstrated in proliferative but not in postconfluent cells. In the latter condition, serum and glucose deprivation triggered autophagy via a transient phosphorylation of ERK1/2, whereas Cd-modified autophagy via a ROS-dependent sustained activation of ERK1/2. Basal autophagy flux in proliferative cells and Cd-induced increases in autophagic markers in postconfluent cells both involved p-ERK1/2. Whether Cd blocks autophagic flux in older cell cultures remains to be clarified but our data suggest dual effects. Our results prompt further studies investigating the consequences that Cd-induced ERK1/2 activation and the related effect on autophagy may have on the intestinal cells, which may accumulate and trap high levels of Cd under some nutritional conditions. Graphical abstract