Expression of bilirubin UDP-glucuronosyltransferase (bUGT) throughout fetal development: intrasplenic transplantation into Gunn rats to correct enzymatic deficiency

Digestive Diseases and Sciences, Vol. 46, No. 12 (December 2001), pp. 2762–2767 (© 2001)

Expression of Bilirubin UDP-Glucuronosyltransferase (bUGT) Throughout Fetal Development Intrasplenic Transplantation into Gunn Rats to Correct Enzymatic Deficiency F.J. CUBERO, BSc,* E. ARZA, BSc,* P. MAGANTO, PhD, M.S.,‡ GARCI´A BARRUTIA, PhD,† N. MULA, MD,‡ A. ORTIZ, PhD,* and R.M. ARAHUETES, PhD*

The aim of this work was to determine the pattern of expression of hepatic bilirubin UDP-glucuronosyltransferase throughout fetal development in rats, with the purpose of using fetal hepatocytes at the most appropiate stage of development for transplantation into Gunn rats lacking bilirubin UDP-glucuronosyltransferase activity and then assessing the therapeutic capacity of the implants. The results show that at day 13 of gestational life there is already bilirubin UDPglucuronosyltransferase gene expression. Twenty-one-day fetal hepatocyte transplantation was also performed into the spleens of hyperbilirubinemic Gunn rats, when ␣-fetoprotein mRNA is still detectable. At 15, 30, and 90 days after transplantation, a mild decrease in total bilirubin serum levels was observed. An increase in bile conjugated bilirubin also was observed at 30 and 90 days. These data suggest the favorable evolution of transplanted cells and show its feasibility for therapy. KEY WORDS: Gunn rats; hyperbilirubinemia; fetal hepatocyte transplantation; bilirubin UDP-glucuronosyltransferase.

Bilirubin is the degradation product of heme, the bulk of which is derived from hemoglobin of senescent erythrocytes and hepatic hemoproteins. Bilirubin is carried in the circulation by binding to plasma albumin. The endothelial lining of the hepatic sinusoids is fenestrated, and the albumin– bilirubin complex enManuscript received April 5, 2001; accepted July 10, 2001. From the *Departamento de Biologı´a Animal II and †Departamento de Biologı´a Celular, Facultad de Biologı´a, UCM, 28040, Madrid, Spain; and ‡Servicio Cirugı´a Experimental, Clı´nica Puerta de Hierro, 28035 Madrid, Spain. This work was financed by grant 08.6/0027.1/98/98 from the Comunidad Auto ´noma de Madrid, Spain. Address for reprint requests: R.M. Arahuetes, Departamento de Biologı´a Animal II, Facultad de Biologı´a, Universidad Complutense, 28040 Madrid, Spain.

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tering the liver through the portal circulation passes through the fenestrae to reach Disse’s space, where bilirubin comes into direct contact with the sinusoidal and basolateral plasma membrane domains of the hepatocyte. Bilirubin, but not albumin, passes into the hepatocyte throughout a specific uptake mechanism, implying that bilirubin must dissociate from albumin before its uptake into the hepatocyte. A fraction of the bilirubin is also derived from catabolism of hepatocellular heme proteins. Storage within the hepatocyte is accomplished by binding of bilirubin to a group of cytosolic proteins, glutathione-S-transferases (also called ligandin or Y-protein). Binding to these proteins keeps bilirubin in solution and inhibits its efflux Digestive Diseases and Sciences, Vol. 46, No. 12 (December 2001)

0163-2116/01/1200-2762/0 © 2001 Plenum Publishing Corporation

EXPRESSION OF bUGT THROUGHOUT DEVELOPMENT

from the cell, thereby increasing net uptake. Conjugation of bilirubin in the endoplasmic reticulum is catalyzed by bilirubin UDP-glucuronosyltransferase (bUGT), forming bilirubin monoglucuronide and diglucuronide. Conjugation is obligatory for efficient transport across the bile canaliculus (1). Although bilirubin is potentially toxic, it is generally rendered harmless because of binding to serum bilirubin and efficient conjugation and excretion by the liver. However, a complete or near-complete deficiency of bUGT activity results in severe disorders such as Crigler-Najjar syndrome type 1 (CN 1), an autosomal recessive disorder characterized by nonhemolytic jaundice resulting from mutations to the gene encoding bUGT. The Gunn rat is an accurate model of this disease because the bUGT gene in this strain carries a premature stop codon (2, 3) leading to the truncation of the carboxy-terminal 150 amino acids of the bUGT and the inactivation of their catalytic function (4). Although orthotopic liver transplantation has become a standard treatment of CN1 (5, 6), fetal hepatocyte transplantation (FHT) into the spleen has been used for the last few years for the treatment of experimental enzyme deficiencies (7–9). In the present study we describe the pattern of expression of bUGT throughout the fetal development with the aim of using FHT at the most ideal stage of development to restore liver function impaired by disease or congenital defect. Furthermore, we tested a E21 FHT to evaluate the changes in bilirubin production. Our results demonstrate that this technique is a promising one. MATERIALS AND METHODS Animals. Female Wistar rats (200 –250 g) with timed pregnancy were bred in the laboratory in solid-floored cages containing wood shavings and standard breeder chow in pellet form. Free access to water was assured from water bottles suspended at the top of the cages. Animals were housed in a climate-controlled (21°C) room with a 12-hr light– dark cycle. Gestational age was determined taking the day of a sperm-positive vaginal smear as day 0. The subseTABLE 1. FETAL WEIGHT Day of development E13–14 E15 (N E17 (N E19 (N E21 (N E22 (N

(N ⫽ 20) ⫽ 25) ⫽ 22) ⫽ 26) ⫽ 30) ⫽ 28)

*Values are mean ⫾

quent rapid weight gain of the dam confirmed the success of the pregnancy. Pregnant rats between 13 and 22 days of gestation (E13–E22) underwent Caesarean section under general anesthesia (ether). Fetuses were removed, and the livers were frozen in liquid N2. Both fetuses and livers were weighed. To test the functionality of intrasplenic transplanted hepatocytes in the Gunn rat (gg), a bearer of a bUGT deficiency, the donors were 21-day syngeneic dominant homozygous GG normal rat fetuses. Rats were killed 7, 15, and 30 days after the implant, and light microscopy was performed on liver and spleen. Bilirubin levels were assessed from blood, which was withdrawn from the jugular vein before the transplant, and at the end of the study, and from bile, withdrawn from the bile duct. Care of these animals complied with the norms stipulated by the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals issued by the National Society for Medical Research and the National Academy of Sciences of the United States, respectively. Preparation of Hepatocytes from Fetal Rat. The 21-day GG fetal hepatocytes were isolated by using the method described by Berry and Friend (10) and modified by Arahuetes et al (8). The number and viability of cells were estimated by trypan blue exclusion. The average hepatocyte yield from fetuses of 21-day gestational age was reported to be 100 ⫻ 106 cells/g of fetal liver. Trypan blue exclusion was 85–90%. Fetal Hepatocyte Transplantation. The recipients were subjected to midline laparotomy under ether anesthesia. Transplantation consisted of direct injection into the red pulp of the spleen of a suspension containing approximately 40 million fetal hepatocytes (viability: 85–95%). Detection of AFP and bUGT mRNAs. Total mRNA was extracted from the livers of Wistar rats with timed pregnancy (E13–E22). The tissue was denatured with 1 ml Ultraspec RNA (Biotecx Laboratories) per 100 mg tissue, and RNA was extracted according to the method of Chirgwin et al (11). Total RNA from the liver of both adult and fetal Wistar rats was used as control. The total RNA was quantified using a GeneQuant RNA/DNA calculator (Pharmacia). Dot Blot was performed to detect the mRNAs of interest. Prehybridization was carried out for 60 min at 65°C using a prehybridization solution that consisted of 5⫻ SSC, 0.1% SDS, 5% dextran sulfate, and blocking fluid A from the Gene Images random prime labelling module kit (Amersham). The probe was added to the prehybridization solu-

LIVER WEIGHT REPRESENTED AS FETAL WEIGHT WEIGHT THROUGHOUT DEVELOPMENT

AND

VS

FETAL LIVER

Fetal weight (g)*

Fetal liver weight (mg)*

Liver weight (%)

0.3 ⫾ 0.05 0.5 ⫾ 0.15 0.7 ⫾ 0.17 2.2 ⫾ 0.19 4.3 ⫾ 0.38 5.9 ⫾ 0.46

5.5 ⫾ 0.2 10.0 ⫾ 3.0 60.0 ⫾ 5.2 220.0 ⫾ 12.0 370.0 ⫾ 33.3 400.0 ⫾ 39.4

1.1 3.3 8.6 10.0 8.6 6.8

SEM.

Digestive Diseases and Sciences, Vol. 46, No. 12 (December 2001)

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CUBERO ET AL

Fig 1. Dot blot for bUGT expression in fetal liver. (A–C) E13 mRNA (0.5, 4, 8 ␮g); (D and E) E14 mRNA (0.5, 4, 8 ␮g); (F and G) total spleen RNA (0.5, 5 ␮g).

tion at a concentration of 10 ng/ml, and the hybridization reaction took place over a period of 16 hr at 65°C. The probes used were the 900-bp pAFP (12) and 414-bp UGT (13). These cDNAs were labeled using the Gene Images random prime labeling module kit (Amersham). For dot blot in this study, 150 ng of both probes were labeled. The solutions used for posthybridization washes were 1⫻ SSC, 0.1% SDS (w/v), and 0.01⫻ SSC, 0.1% SDS (w/v). Detection of the albumin mRNA was carried out by means of the Gene Images CDP-Star detection module Kit (Amersham). Quantification of AFP. Quantification of AFP levels in amniotic fluid, and in E20 and E21 fetal serum and maternal serum was assessed by means of ELFA (enzyme-linked fluorescent assay) using the automatic VIDAS AFP test (Biomerioux). Measurement of Bilirubin Levels. Bile and serum bilirubin levels were measured using a clinical kit obtained from Sigma Co. (St. Louis, Missouri, USA). Statistical Analysis. After testing for normality and homogeneity of variances, Student’s t test was used for statistical comparison of values expressed as mean ⫾ SD. A value of P ⬍ 0.05 was considered to be significant.

RESULTS Relationship Between Body Weight, Liver Weight, and Day of Development. As shown in Table 1, fetal weight and fetal liver weight increase throughout development. The percentage of liver weight starts to increase between days E15–E16 reaching its maximum by E19. From E19 onwards, this ratio drops due to the growth of the rest of the body’s organs. Bilirubin UDP-Glucuronosyltransferase mRNAs. The average yield in the isolation of total RNA was 218 ␮g/ml of tissue, with a 260/280 nm ratio ranging 2.0. Dot blot analysis of the bUGT gene expression in E13 and E14 samples of fetal livers resulted in a positive signal in both samples, whereas total RNA from commercial spleens were always negative for bUGT mRNA (Figure 1). Dot blot study of the bUGT gene expression in E15, E17, E19, and E21

Fig 2. Dot blot for bUGT expression in fetal liver. (A and B) E15 mRNA (0.5 and 4 ␮g); (C and D) E16 mRNA (0.5 and 4 ␮g); (E and F) E17 mRNA (0.5 and 4 ␮g); (G and H) E21 mRNA (0.5 and 4 ␮g); (I and J) E22 mRNA (0.5 and 4 ␮g); (K–P) total liver RNA (0.1– 4 ␮g).

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Digestive Diseases and Sciences, Vol. 46, No. 12 (December 2001)

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Fig 3. Dot blot for AFP mRNA in fetal liver. (A–C) E21 total RNA (1 ␮g).

samples of fetal livers resulted in a positive signal in all samples. In the control group (K-P), total adult liver RNA was also positive for bUGT mRNA (Figure 2). AFP mRNAs. The average yield of total RNA isolated was 1.882 ␮g/ml of tissue, with a 260/280 nm ratio ranging 0.94. Dot blot study of AFP gene expression in E21 resulted in a positive signal in the sample (Figure 3). Quantification of AFP. As shown in Table 2, levels of the enzyme start to decrease from E21 both in fetal and maternal serum as well as in amniotic fluid compared to E20. This marked decrease is as much as 50% in amniotic fluid. Measurement of Bilirubin. In serum, basal values of total bilirubin were measured in the rats before FHT. These data were compared to those in the same Gunn rats at 15, 30, and 90 days after FHT. Differences were significant between the basal values and the total bilirubin levels at all days after transplantation (Table 4). These results suggest the possible role of fetal hepatocyte transplantation. As mentioned before, bile was withdrawn from the bile duct. Bile was obtained through a catheter and the animals subsequently died. This is the reason why basal data of conjugated, unconjugated, and total bilirubin of nontransplanted animals were compared with those in transplanted Gunn rats. In normal rats, conjugated bilirubin represents the major form of bilirubin in bile, reaching 85% of the total bilirubin. In contrast, Gunn rats, incapable of conjugating bilirubin, have more nonconjugated bilirubin in bile (around 55% of the total bilirubin). TABLE 2. AFP LEVELS IN AMNIOTIC FLUID AND FETAL MATERNAL SERUM AT E21 AND E22*

AND

Day of gestation

Fetal serum

Maternal serum

Amniotic fluid

E20 E21

24.18 ⫾ 1.21 19.79 ⫾ 3.16

22.27 ⫾ 3.12 18.93 ⫾ 3.63

31.17 ⫾ 5.70 15.49 ⫾ 2.43

*Values are mean ⫾

SEM.

Digestive Diseases and Sciences, Vol. 46, No. 12 (December 2001)

Fig 4. Levels of AFP in maternal and fetal serum in amniotic fluid (ng/ml).

After FHT into Gunn rats, conjugated bilirubin becomes the major form of bilirubin in bile by day 30 after transplantation. At 90 days after transplantation, this percentage is even greater (around 70%) and tends to ressemble the levels of bile bilirubin in normal rats (Table 3). DISCUSSION The liver is the largest organ in the human body and in adults represents around 2.5% of the total body weight. In the rat, the liver weight can multiply threefold between E16 and E22, whereas the ratio of liver to body weight decreases as fetal development continues in most mammals (14). The evolution of the weight gain (Table 1) throughout gestation and even the early neonatal period is relevant. This ratio increases from E13 and reaches its maximum by E19. The physiological meaning of this event may be justified by the growth and proliferation of the hepatic parenchyma as well as the large hemopoiesis in this stage. After the strong increase in fetal body weight, accompanied by the growth of the organs, the weight change of the liver starts to decrease by E19 and stabilizes after birth. In the rat liver, AFP mRNA appears at the time of the formation of the liver primordium by E10.5. AFP is immunocytochemically detectable at E11.5. Albumin starts to be expressed just one day later than AFP, which is coincident with the commitment of endodermal cells to the lineage of liver cells (15). AFP levels in amniotic fluid, fetal serum, and maternal serum at E20 and E21 indicate that AFP dramatically diminishes (Figure 4) as liver maturation proceeds (16). These data suggest that at E21 the

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CUBERO ET AL TABLE 3. TOTAL, CONJUGATED, AND NONCONJUGATED BILIRUBIN IN BILE*

Bilirubin in bile (mg/dl)

Gunn rats (gg) (N ⫽ 5) AV

RV

30 days post-FHT (N ⫽ 4) AV

90 days post-FHT (N ⫽ 4) RV

AV

RV

2.24 ⫾ 0.31 100 1.00 ⫾ 0.00 100 1.21 ⫾ 0.24 100 1.23 ⫾ 0.18 55 0.40 ⫾ 0.20 40 0.53 ⫾ 0.10 34 1.01 ⫾ 0.14 45 0.60 ⫾ 0.20 60 0.80 ⫾ 0.31 66

Bt Bl Bc

*Levels in Gunn rats, 30 and 90 days after transplantation and normal rats. AV: absolute value (mean ⫾ SEM) RV: relative value.

hepatocyte is differentiated and therefore suitable for transplantation. Bilirubin UDP-glucuronosyltransferase mRNA expression is detectable by dot blot at E13. Our results differ from those of Huang et al (17), who detected expression at E17, using the same probe. This finding is evidenced by the fact that at E13 most fetal hepatoblasts are bipotential, being capable of differentiation towards either the hepatocytic or the bile duct lineages (18). At E19 an immature activity of the bUGT was detected in vitro by Fevery et al (19). This activity gradually increases until birth, when it dramatically reaches half of adult levels by postnatal day 1. Adult levels are reached by postnatal day 4. These findings are consistent with the hypothesis that bUGT activity of fetal liver in utero remains suppressed for at least two reasons: (1) the fetal liver is exposed to high levels of gestational hormones; and (2) the placenta efficiently clears bilirubin formed by the fetus in utero so that the circulating concentration of this potential inducer remains low. The rapid development of bUGT activity neonatally would also be consistent with the above hypothesis. At birth, the neonatal pup excretes the transplacentally infused gestational hormones once it is separated from the placental circuTABLE 4. LEVELS

OF

TOTAL SERUM BILIRUBIN*

Total serum bilirubin (mg/dl)

Pre-FHT 6.39 ⫾ 2.56 6.65 ⫾ 0.83 7.33 ⫾ 1.52

15 days post-FHT (N ⫽ 4) 5.03 ⫾ 2.44†

30 days post-FHT (N ⫽ 4) 3.69 ⫾ 0.82†

90 days post-FHT (N ⫽ 4)

5.64 ⫾ 1.18†

*Pre-FHT values were compared to those in the same subjects at 15, 30 and 90 days post-FHT. Levels were significant (P ⬍ 0.05) in all groups (pre-FHT-15 days post-FHT; preFHT-30 days postFHT; pre-FHT-90 days post-FHT). †P ⬍ 0.05.

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lation. Simultaneously, the animal’s endogenously produced bilirubin serves to induce bUGT activity for its hepatic clearance (20). The rapid development of bUGT activity neonatally suggests that bUGT expression ocurrs before birth. In humans, the newborn liver is incapable for capture, conjugation, and clearance of bilirubin. This physiological hyperbilirubinemia lasts for the first days after birth. Bilirubin levels rapidly decline to the adult levels, which suggests that induction of bUGT mRNA traduction has started. In this work we assessed a preliminary E21 fetal hepatocyte transplantation into hyperbilirubinemic Gunn rats to test the evolution of the liver cells in the receptors. Furthermore, it has previously been demonstrated by our group that immediately after transplantation the fetal hepatocytes migrated to the liver via the portal vein. They observed clusters of hepatocytes in the small branches of the portal vein up to 15 days after fetal hepatocyte transplantation (9). Our results show that after E21, FHT total serum bilirubin levels tend to decrease. This is concomitant with the increase of bile conjugated levels. These findings suggest a possible therapeutic role of fetal hepatocyte transplantation in the receptor. According to these conclusions and, with a view to using fetal hepatocytes in the most appropiate stage of development and their transplantation to correct genetic metabolopathies in an experimental model of hyperbilirubinemia, the Gunn rat, further studies on the usage of fetal hepatocytes in early stages of the hepatic ontogeny are necessary. Furthermore, these hepatoblasts keep all their potentialities and characteristics of low immunogeneicity and high proliferativity. ACKNOWLEDGMENTS We wish to thank Dr. Sellem and Dr. Sato for the probes for AFP and bUGT, respectively; Dr. Isabel Millan for the statistical analysis; Ms V. Dixon for her editorial assistance; and the Animalary of the Faculty of Biology for the breeding and handling of the Wistar and Gunn rats.

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