Sn-protoporphyrin blocks the increase in serum bilirubin levels that develops postnatally in homozygous Gunn rats.

Administration of Sn-protoporphyrin to Gunn rats that are characterized by a genetically determined absence of UDP-glucuronyl transferase activity for bilirubin, 24-30 h after birth, prevented the marked increase in serum bilirubin concentration that occurs in these animals in the postnatal period. A second administration of Sn-protoporphyrin at day 6 maintained serum bilirubin levels in the neonates at the initial level for an additional 6 d. In contrast, in untreated Gunn neonates, serum bilirubin levels increased substantially as expected during the immediate 2-wk period after birth. Studies in adult Gunn rats demonstrated that Sn-protoporphyrin administration diminished biliary bilirubin output, decreased tissue heme oxygenase activity, and did not alter hepatic cytochrome P450 levels. These findings raise the possibility that Sn-protoporphyrin may prove clinically useful in maintaining low levels of serum bilirubin in congenitally jaundiced individuals, such as patients with the Crigler-Najjar syndrome.

Takashi Iyanagi: UGT1 gene complex: from Gunn rat to human.

UDP-glucuronosyltransferases (UGT) comprise a large gene superfamily that can be classified, based on the degree of amino-acid similarity between isoforms, into several gene families. Among these gene families, the UDP-glucuronosyltransferase family 1 (UGT1) gene is a unique gene complex organized to generate enzymes that share a common carboxyl terminal portion and are unique in the variable amino terminal region. Each variable exon I is preceded by a regulatory 5'-region and, in response to a specific signal, transcription processing splices mRNA from each unique exon 1 to the four common exons ( 2, 3, 4, and 5) to provide a template for synthesis of the individual isoforms. A novel clue to elucidate the gene structure of mammalian UGT1 was cDNA cloning of rat UGT1A6 from the hyperbilirubinemic Gunn rat by Professor Takashi Iyanagi Ph.D. The elucidation of the structure of the rat UGT1 gene complex has led to a greater understanding of the genetic basis of Crigler-Najjar and Gilbert's syndromes. Now, examination of the UGT1 gene structure in hyperbilirubinemic patients has revealed more than 100 different genetic defects in Crigler-Najjar syndromes and one genetic alternation that accounts for the majority of Gilbert's syndrome cases. This review of a chapter in UGT history will focus on the extensive research of Iyanagi and coworkers with the rat UGT1 gene complex and advancing to the study of the human gene.

Evidence against an abnormal hepatic microsomal lipid matrix as the primary genetic defect in the jaundiced Gunn rat.

The congenitally jaundiced Gunn rat does not conjugate bilirubin but does conjugate bilirubin dimethyl diester. Partial defects in conjugating p-nitrophenol and demethylating aminopyrine are also evident. A proposed mechanism to explain this combination of findings is a defective microsomal membrane. To examine the 'matrix' of Gunn microsomal membranes, hepatic microsomes were isolated from Gunn (jj) and outbred Wistar (JJ) rats and were studied by electron paramagnetic resonance spectroscopy of 7-doxylstearic and 12-doxylstearic acid probes, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, glucose-6-phosphatase activity vs. temperature, and lipid analysis. The data indicate several factors related to lipid bilayer order do not differ in microsomes from jj and JJ.

The genesis and ultrastructural appearance of spontaneous renal lesions in the homozygous Gunn rat.

Deposition of bilirubin at the tip of the renal papilla has been studied in the infant homozygous Gunn rat. The deposits form during the 4th wk of life in ground substance along basement membranes of capillaries and tubules and along the borders of interstitial cells. Deposits also form in capillary lumens. The early ultrastructural appearances indicate that unconjugated bilirubin is not directly cytotoxic. This conclusion has important implications for the pathogenesis of enhanced nephrotoxicity in the homozygous Gunn rat and ultimately for the pathogenesis of analgesic nephropathy.

The congenic normal R/APfd and jaundiced R/APfd-j/j rat strains: a new animal model of hereditary non-haemolytic unconjugated hyperbilirubinaemia due to defective bilirubin conjugation.

In this paper the production of the R/APfd-j/j strain which is congenic with the R/APfd strain is reported. The R/APfd-j/j completely lacks hepatic bilirubin UDP-glucuronyltransferase activity, as do our GUNNXR/Pfd-j/j rat strain and various other stocks of GUNN rats (j/j) described in the literature. Our recombinant inbred strain GUNNXR/Pfd-j/j was produced from non-inbred GUNN (j/j) rats. This GUNNXR/Pfd-j/j rat was used as a donor of the jaundice gene j, the R/APfd rat serving as the recipient. After eight backcross-intercross cycles (16 generations) the R/APfd-j/j strain was obtained which is congenic with the R/APfd strain. Congenicity was demonstrated by various techniques including transplantation of skin tissue, strain-specific tumour cells and hepatocytes, the mixed lymphocyte reaction, and comparison of biochemical markers. The potential of the novel inbred strain of jaundiced rat, R/APfd-j/j, and the corresponding control strain R/APfd for biochemical and clinical studies of bilirubin metabolism are briefly discussed.

Transfer of the heritable nonhemolytic jaundice trait from the Gunn rat to the Sprague-Dawley rat.

Congenital nonhemolytic jaundice as observed in the Gunn rat was transferred successfully to the Sprague-Dawley rat. This jaundice trait occurs as the result of a deficiency of bilirubin glucuronyltransferase and appeared to transfer by simple Mendelian inheritance. A comparison of jaundiced Gunn with jaundiced Gunn-Sprague-Dawley cross rats for plasma bilirubin level, bilirubin glucuronyltransferase activity and female reproductive performance showed no significant difference between the two jaundiced rat groups. The phenotypic expression of the jaundice trait as viewed by the parameters used in this study appeared to be the same for both the Gunn and Gunn-Sprague-Dawley cross rats. The transfer of the jaundice trait to another rat strain enhances the opportunity to characterize this animal model and to determine the possible influence of a long-term closed mating system.

Effect of blue light on the urinary excretion of tryptophan metabolites in adult heterozygous and homozygous Gunn rats.

Tryptophan metabolism along the kynurenine pathway has been studied in male and female heterozygous and homozygous adult Gunn rats after load of amino acid before and after exposure to blue light. The percentage of tryptophan metabolites in each group of rats was slightly higher in females than in males. Before irradiation the excretion of metabolites was larger in male and female heterozygous groups than the corresponding groups of the homozygotes. After irradiation only the groups of male and female heterozygous Gunn rats showed a decrease in the total excretion of the metabolites. Therefore blue light exposure dose not seem to influence the metabolism of tryptophan in homozygous Gunn rats.

A new mutant strain of Gunn-LA Wistar rats with genetic deficiencies in bilirubin and androsterone uridine diphosphate-glucuronosyltransferases.

Hooded Gunn rats which have low-activity of 4-nitrophenol uridine diphosphate-glucuronosyltransferase (GT) and defect in bilirubin GT were crossed with albino LA Wistar rats with a defect in androsterone GT. From F2 and F3 progeny were selected Gunn-LA Wistar rats which have defective bilirubin and androsterone GTs and low-active 4-nitrophenol GT. These rats had either hooded or albino coat color. In order to establish a uniform genetic background, albino male Gunn-LA Wistar rats were crossed with heterozygous albino female Gunn-LA Wistar rats in terms of bilirubin GT. The offsprings were all albino and were classified to Gunn-LA Wistar rats and heterozygous Gunn-LA Wistar rats by assaying their hepatic GT activities or by the appearance of jaundice. The linkage relationships between GT and coat color genes are discussed.

Accumulation of cholesteryl esters associated with cerebellar hypoplasia in jaundiced Gunn rats.

A marked accumulation of cholesteryl esters in the cerebellum of 20-day-old jaundiced homozygous Gunn rats was shown by quantitative TLC analyses and it was larger than that found in heterozygotes.

Rates of protein synthesis and degradation in Gunn rat cerebellum with bilirubin-induced cerebellar hypoplasia.

The rates of protein synthesis and degradation, and the endogenous leucine content on days 13 and 30, were examined in the cerebellum of homozygous Gunn rats with cerebellar hypoplasia and compared with those of heterozygotes. The rate of protein synthesis based on milligrams of protein was significantly lower on day 30 and the degradation rate was about ten times faster between days 13 and 18. Furthermore, leucine concentration was about 1.7 times higher on day 13. The present results suggest that the lowered protein concentration previously observed in the cerebellum of homozygous Gunn rats is ascribed to the increased rate of protein degradation on day 13, and to the decreased rate of protein synthesis on day 30.

Enzyme activities of tryptophan metabolism in heterozygous and homozygous Gunn rats before and after blue light exposure.

The effect of light exposure in the enzyme activities, involved in the tryptophan metabolism along the kynurenine pathway, has been studied in male and female adult hetero-and homozygous Gunn rats. Light exposure did not seem to influence these enzyme activities. The groups of non-icteric heterozygotes however showed a higher activity of liver tryptophan pyrrolase than in the groups of icteric homozygotes. Kynureninase activity was similar in all the groups of rats, whereas liver kynurenine aminotransferase activity appeared slightly higher in the groups of male rats with respect to female rats, but no difference existed before and after irradiation. In addition the male heterozygotes showed a higher kynurenine aminotransferase activity in kidneys than the other groups of rats.

Vitamin A (retinol) status in the Gunn rat. The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Vitamin A (retinol) status and the effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 10 micrograms X kg-1, on vitamin A in the liver and serum, and on the hepatic UDP-glucuronosyltransferase (UDPGT) (EC 2.4.1.17) activity, were studied in heterozygous (GW) and homozygous (GG) Gunn rats. 1) Data from vitamin A analyses demonstrate that the amount of vitamin A stored in the liver of untreated Gunn rats is of the same magnitude as that of Sprague-Dawley rats. 2) The retinol content in the liver of both GG and GW rats was reduced to about 50% by TCDD-treatment. 3) Retinol levels in serum were found to be variable and no significant effect due to TCDD could be observed. 4) No correlation between the TCDD-induced reduction of vitamin A and the induction of UDPGT activity by TCDD could be demonstrated in this study. The vitamin A reduction caused by TCDD was considerably less in the Gunn rat than in the Sprague-Dawley rat, and the results indicate that the Gunn rat is more resistant to TCDD than other strains of rat. TCDD-induced reduction of liver vitamin A seems to some extent to correlate with TCDD-toxicity in different strains of rat. The specific properties of the Gunn rat and its relatively high resistance to TCDD make it a valuable tool in studies about the mechanism of TCDD-toxicity.

Molecular basis of multiple UDP-glucuronosyltransferase isoenzyme deficiencies in the hyperbilirubinemic rat (Gunn rat).

The Gunn rat is a mutant strain of Wistar rat which has unconjugated hyperbilirubinemia as a result of the absence of hepatic UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. The Gunn rat is also deficient in UDPGT activities toward phenol substrates, and also toward digitoxigenin-monodigitoxiside. We have demonstrated that the defect of the isoenzyme for 4-nitrophenol (4NP) in Gunn rat liver arises from a -1 frameshift mutation that removes 115 amino acids from the COOH terminus (Iyanagi, T., Watanabe, T., and Uchiyama, Y. (1989) J. Biol. Chem. 264, 21302-21307). To investigate the molecular basis of defects in other UDPGT isoenzymes, we isolated and sequenced cDNAs from a Gunn rat liver library using mutant 4NP-UDPGT cDNA as a probe. Three novel cDNAs were identified that had identical 3'-regions of 1362 base pairs containing a single-base deletion in the same position as that of the mutant 4NP-UDPGT cDNA. However, their 5'-regions, encoding the substrate-binding domain, showed no more than 40% homology to that of 4NP-UDPGT. These data provide evidence that defects in some UDPGT isoenzymes in the Gunn rat are caused by a single mutation that results in the formation of a common truncated COOH terminus. Furthermore, the data also suggest that these mRNAs are transcribed from a single gene and that the 5'-exons are transcribed independently and differentially spliced to common 3'-exons encoding the conserved domain.