Galactosamine hepatitis: Key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

https://www.pnas.org/content/pnas/93/18/9565.full.pdf

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice

Tumor necrosis factor is a terminal mediator in galactosamine/endotoxin-induced hepatitis in mice.

The effects of d-galactosamine, d-glucosamine, and 2-deoxy-d-galactose on rat liver uracil nucleotides were studied in vivo. Enzymic and isotope dilution analyses of the UDP-sugars and of uridine phosphates revealed three major, related changes: an accumulation of the respective UDP-sugar derivatives, a marked decrease of UTP, UDP, and UMP, and a subsequent increase of the sum of hepatic uracil nucleotides. The decrease of uridine phosphates was accompanied by diminished contents of UDPG and UDP-galactose. UMP of total liver RNA was not altered significantly. Inhibition of uridylate synthesis by use of 6-azauridine resulted in a suppression of the d-galactosamine-induced stimulation of uridine phosphate synthesis and of the increase in total acid soluble uracil nucleotides. The trapping of uridine phosphates by formation of UDP-sugar derivatives was most pronounced and most prolonged after administration of d-galactosamine. The uridine phosphate content was lowered to less than 10% of normal within three hours, while the sum of uracil nucleotides increased by 0.35 μmole x g liver−1 x hour−1, from an initial value of 1.24 μmole/g. The quantitative analysis of the time dependent changes of UDP-hexosamines, UDP-N-acetylhexosamines, UDPG, and UDP-galactose revealed a pronounced alteration by d-galactosamine of the UDP-sugar pattern.



Corresponding changes in the distribution of liver uracil nucleotides were obtained after administration of d-glucosamine and 2-deoxy-d-galactose. Both, however, are ineffective in provoking hepatitis. In contrast to d-galactosamine, d-glucosamine and 2-deoxy-d-galactose do not lead to the formation of UDP-hexosamines; furthermore they are less efficient in trapping uridine phosphates in vivo. These observations contribute to an understanding of the orotate-mediated prevention of the galactosamine-induced liver damage, and of the role of pyrimidine nucleotides in this experimental hepatitis.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1970.tb01160.x

The Trapping of Uridine Phosphates by d-Galactosamine, d-Glucosamine, and 2-Deoxy-d-galactose

1H NMR spectroscopy of urine combined with pattern recognition (PR) methods of data analysis has been used to investigate the time-related biochemical changes induced in Sprague-Dawley rats by three model hepatotoxins: alpha-naphthyl isothiocyanate (ANIT), d-(+)-galactosamine (GalN), and butylated hydroxytoluene (BHT). The development of hepatic lesions was monitored by conventional plasma analysis and liver histopathology. Urine was collected continuously postdosing up to 144 h and analyzed by 600-MHz 1H NMR spectroscopy. NMR spectra of the urine samples showed a number of time-dependent perturbations of endogenous metabolite levels that were characteristic for each hepatotoxin. Biochemical changes common to all three hepatotoxins included a reduction in the urinary excretion of citrate and 2-oxoglutarate and an increased excretion of taurine and creatine. Increased urinary excretion of betaine, urocanic acid, tyrosine, threonine, and glutamate was characteristic of GalN toxicity. Both GalN and ANIT caused increased urinary excretion of bile acids, while glycosuria was evident in BHT- and ANIT-treated rats. Data reduction of the NMR spectra into 256 integrated regions was used to further analyze the data. Mean values of each integrated region were analyzed by principal components analysis (PCA). Each toxin gave a unique time-related metabolic trajectory that could be visualized in two-dimensional PCA maps and in which the maximum distance from the control point corresponded to the time of greatest cellular injury (confirmed by conventional toxicological tests). Thereafter, the metabolic trajectories changed direction and moved back toward the control region of the PR map during the postdose recovery phase. The combination of urinary metabolites which were significantly altered at various time points allowed for differentiation between biliary and parenchymal injury. This NMR-PR approach to the noninvasive detection of liver lesions will be of value in furthering the understanding of hepatotoxic mechanisms and assisting in the discovery of novel biomarkers of hepatotoxicity.

Nuclear Magnetic Resonance Spectroscopic and Principal Components Analysis Investigations into Biochemical Effects of Three Model Hepatotoxins

d-Galactosamine has been shown to produce a liver damage closely related to human viral hepatitis. d-Galactosamine-1-phosphate and UDP-galactosamine were identified as the predominant early metabolites of galactosamine in rat liver. The conversion of galactosamine-1-phosphate to UDP-galactosamine is shown to be catalyzed by UDP-glucose: α-d-galactose-1-phosphate uridylyltransferase. The low affinity of this enzyme for galactosamine-1-phosphate explains in part the high levels of this compound found in galactosamine treated livers. Under these conditions galactosamine-1-phosphate accumulation is enhanced by the strongly reduced levels of UDPG. Galactosamine-1-phosphate inhibits the UDPG-pyrophosphorylase reaction, the type of inhibition being mainly competitive with glucose-1-phosphate. In the presence of the concentrations of galactosamine-1-phosphate and glucose-1-phosphate found in vivo after galactosamine treatment, UDPG-pyrophosphorylases from rat and calf liver are strongly inhibited in vitro. By these mechanisms galactosamine-1-phosphate counteracts its own conversion to UDP-galactosamine. The influence of the strongly diminished UDPG levels on the UDPG-linked syntheses of glycogen, heteropolysaccharides and glucuronides as well as the trapping of uridine phosphates by formation of UDP-hexosamines may play an important role in the induction of galactosamine hepatitis.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1969.tb00677.x

D. Keppler

Papers

Studies on the Mechanism of Galactosamine Hepatitis: Accumulation of Galactosamine‐1‐Phosphate and its Inhibition of UDP‐Glucose Pyrophosphorylase