The anti-mutagenic and antioxidant effects of bile pigments in the Ames Salmonella test

The aim of this study was to explore the potential pro- and anti-mutagenic effects of endogenous bile pigments unconjugated bilirubin (BR), biliverdin (BV) and a synthetic, water soluble conjugate, bilirubin ditaurate (BRT) in the Ames Salmonella test. The bile pigments were tested over a wide concentration range (0.01-2 micromol/plate) in the presence of three bacterial strains (TA98, TA100, TA102). A variety of mutagens including benzo[alpha]pyrene (B[alpha]P), 2,4,7 trinitrofluorenone (TNFone), 2-aminofluorene (2-AF), sodium azide (NaN(3)) and tertiary-butyl hydroperoxide (t-BuOOH), were used to promote the formation of mutant revertants. Tests were conducted with (B[alpha]P, 2-AF, t-BuOOH) and without (TNFone, NaN(3), t-BuOOH) metabolic activation incorporating the addition of the microsomal liver preparation, S9. The bile pigments alone did not induce mutagenicity in any of the strains tested (p>0.05). Anti-mutagenic effects of the bile pigments were observed in the presence of all mutagens except for NaN(3) and the anti-mutagenic effects appeared independent of the strain tested. For TNFone induced genotoxicity, the order of effectiveness was BR> or =BRT>BV. However, the order was BV> or =BRT> or =BR for 2-AF. Antioxidant testing in the TA102 strain revealed bile pigments could effectively inhibit the genotoxic effect of t-BuOOH induced oxidative stress. The apparent antioxidant and anti-mutagenic behaviour of bile pigments further suggests their presence in biological systems is of possible physiological importance.     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

Biliary bilirubin in rats and exposure to metals

After cannulation the bile duct of unexposed rats, changes in bilirubin concentration in the samples of bile collected were observed. During the first hour after the cannulation the bilirubin concentration decreased remaining constant until about the 3.-4. hour and then continuously increased upto the 24. hour when the experiment ended. The final concentration was about 4 fold higher than between the 1.-4. hour after the cannulation. This could explain a large variation in values reported by various authors. This fact significantly complicated the studies on effect of exposure to heavy metal salts on changes of bilirubin concentration in bile and relationship among bilirubin concentrations in the bile fractions 1, 2 and 3. In the bile of unexposed rats comparable bilirubin concentrations were found in the bile fractions 1 and 2. The preliminary experiment with HgCl2 application to rats revealed that in the fraction 3 might be a higher bilirubin concentration than in the fractions 1 and 2, the latter ones being lower than in the bile of unexposed rats. The hypothesis was suggested that the bile fraction 3 was composed from yellow pigments liberated from compounds forming the fractions 1 and 2 with additional amount of pigments originated during disturbed protoporphyrin biosynthesis of their degradation by the metals.     

Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile.

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.     

Effect of biliverdin and bilirubin on the hepatic excretion of bile pigments in the rabbit

1. A study has been carried out on the effect of i.v. infusion of biliverdin and bilirubin at four different dose strengths in sodium pentobarbital-anaesthetized rabbits. 2. Both pigments show similar values for the maximum excretion rate in bile. 3. At doses of 0.45 and 0.60 mg/kg/min, the infusion of biliverdin does not affect bile flow in the resting state, contrary to the negative effect on flow induced by bilirubin. 4. This negative effect induced by bilirubin on bile flow is explained on the basis of its inhibitory action on the bile salt independent fraction (BSIF).     

High-performance liquid chromatographic separation of bilirubin conjugates

A fast sensitive method for the isolation and quantitation of biliary bile pigments by reverse-phase high-performance liquid chromatography has been developed. Nine conjugates of bilirubin as well as unconjugated bilirubin and an internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution. The following sequence of eluted compounds was chemically identified by separating their ethyl anthranilate derivatives by thin-layer chromatography and by their enzymatic formation with UDP-bilirubin transferase and cosubstrate: bilirubin diglucuronide, bilirubin monoglucuronide monoglucoside, bilirubin monoglucuronide monoxyloside, bilirubin monoglucuronide (C-8, C-12), bilirubin diglucoside, bilirubin monoglucoside monoxyloside, bilirubin dixyloside, bilirubin monoglucoside (C-8, C-12), and bilirubin monoxyloside. The use of the commercially available mesobilirubin IX alpha as an internal standard was found to facilitate quantitation of the bilirubin conjugates.     

Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography.

1. Conjugated bile pigments, separated in two fractions by semi-quantitative t.l.c. performed on silicic acid with phenol/water as the developing solvent, were treated with diazotized ethyl anthranilate. Resulting dipyrrylazo derivatives were analysed by quantitative t.l.c. 2. The tentative structure elucidation of tetrapyrrolic bilirubin conjugates and semi-quantitative evaluation of rat bile, post-obstructive human bile and dog bile composition is presented. 3. Homogeneous and mixed hexuronic acid diesters of bilirubin containing glucuronic acid constitute 51% of the total conjugates in normal rat bile, 45% of those in human post-obstructive bile and 38% of those in obstructed rat biles. 4. Monoconjugated bilirubin amounts to 33% of total conjugated bile pigments in normal rat bile, and 17 and 14% in post-obstructive hepatic human bile and gall-bladder bile of dog respectively. After loading with unconjugated bilirubin a greater amount of monoconjugates (56%) occur in the rat bile, whereas bilirubin diglucuronide excretion is decreased (34%). 5. In gall-bladder bile of normal dog, 40% of glucose-containing diconjugates, 32% of homogeneous and/or mixed hexuronic acid (mainly glucuronic acid) diesters of bilirubin and 14% of xylose-containing diconjugates are estimated. 6. Increased amounts of bilirubin conjugates, including some with unidentified uronic acid groups, were observed in cholestatic rat biles and quantities of conjugates with glucuronic acid were decreased.     

Bile Pigments in the Bile of Freshwater Fish,Rainbow Trout,Tilapia, and Pejerrey

Bile pigments in the bile of the rainbow trout, Salmo gairdneri, tilapia, Tilapia nilotica, and pejerrey, Odonthetes bonariensis, were analyzed by HPLC and TLC.

Major bile pigments of rainbow trout and pejerrey were bilirubin glucuronides and ditauro-bilirubin, respectively. Ditaurobilirubin was not detected in rainbow trout and bilirubin glucuronides were scarcely found in pejerrey. Biliverdin seemed to be the sole bile pigment in tilapia, but it was a minor component in the other fish. These results are in accord with the previous reports in which the diversity of bile pigment composition was demonstrated in some fish.     

Triamcinolone activation of renal ammonia production.

The effects of scillaren and dinitrophenol on bilirubin excretion by the perfused rat liver were studied. Both compounds inhibited bile flow, scillaren by 20 to 40%, and dinitrophenol by 60 to 80%. Bilirubin excretion was also impaired. However, the effect of scillaren on bilirubin excretion was less than that on bile flow, as indicated by an increase in the bile bilirubin concentration, whereas dinitrophenol had a greater effect on bilirubin excretion than on bile flow. Dinitrophenol also inhibited the hepatic removal of unconjugated bilirubin from the perfusate, probably because it impaired the initial uptake and/or storage of unconjugated bilirubin by the perfused liver.     

Changes in bilirubins in human prenatal development.

1. A densitometric method has been developed for the quantification of azodipyrroles derived from dog bile pigments treated with diazotized ethyl anthranilate. 2. This method was used to estimate the bilirubins in bile and meconium from foetuses of 14-36 weeks gestation. 3. The proportion of the bilirubins in foetal bile changed during gestation. (a) No bile pigments were found until 14 weeks. (b) Between 14 and 15 weeks bilirubin-IX beta was the only bile pigment detected. (c) At 16-17 weeks some unconjugated bilirubin-IX alpha was found in the bile, but up to 20 weeks bilirubin-IX beta was the predominant bilirubin in the bile. (d) At about 20 weeks glucose, xylose, and an unidentified bilirubin-IX alpha monoconjugate were found in the bile. (e) Between 20 and 23 weeks bilirubin-IX alpha glucuronide appeared in the bile. (f) At 30 weeks monoconjugates of bilirubin-IX alpha were the predominant bilirubins in the bile. (g) Only in full-term foetuses was bilirubin-IX alpha monoglucuronide the major bilirubin derivative.     

Synergistic interaction between vitamin E and the bile pigments bilirubin and biliverdin

The oxidation of soybean phosphatidylcholine (PC) liposomes initiated with a lipid-soluble azo compound within the liposomal membranes has been studied in the absence and presence of membrane-bound vitamin E and water-soluble bile pigments. In the absence of vitamin E, lipid peroxidation proceeded linearly and without delay. Low micromolar amounts of bilirubin ditaurine (BR-DT, a model compound of conjugated bilirubin) or biliverdin (BV) inhibited the oxidation of PC significantly and in a concentration-dependent way. In contrast, neither taurine, ascorbic acid nor reduced glutathione inhibited significantly under these conditions. Both bile pigments were consumed during their protective action. Vitamin E incorporated into the liposomal membranes suppressed the oxidation initially almost completely, thereby producing an induction period. In the combined presence of vitamin E and either of the two bile pigments at 10 microM each, this induction period was increased by at least 200%. In contrast, when 10 microM vitamin E was combined with an equimolar concentration of reduced glutathione, the induction period increased by only about 30%. BR-DT and BV both spared the consumption of vitamin E during the oxidation of PC liposomes. These results demonstrate that conjugated bilirubin and BV located in the aqueous phase can directly scavenge lipid radicals to some extent. Furthermore, both bile pigments can act synergistically with membrane-bound vitamin E to prevent lipid peroxidation initiated in the lipid phase, most likely through regeneration of the vitamin from its chromanoxyl radical.     

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea)

Summary Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of³H-biliverdin were detected in hepatic bile following the intravenous administration of³H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.     

Assay of beta-glucuronidase in bile following ion-pair extraction of pigments and bile acids

A method for improving the assay of beta-glucuronidase in hepatic and gallbladder bile is described. The method uses ion-pair extraction with N,N,N-triheptyl-1-heptanaminium bromide to remove pigments and bile acids. Conjugated bilirubin, unconjugated bilirubin, and taurine and glycine conjugates of deoxycholic and chenodeoxycholic acids are extracted efficiently from bile by the procedure. The sensitivity of the spectrophotometric assay of beta-glucuronidase in bile using phenolphthalein glucuronide is increased significantly.     

Experimental Research on the In Vitro Antitumor Effects of Crataegus sanguinea

Crataegus sanguinea is a wild plant, which has been widely grown in the north and south of the Tianshan mountains in Xinjiang. In order to explore their anti-cancer properties, edible wild plants from Xinjiang have been tested for their antitumor properties. We used Ames tests, mouse bone marrow polychromatic erythrocytes micronucleus tests, and tumor cells cultured in vitro to study the anti-mutagenic and anti-tumor effects of C. sanguinea extract. We have shown that C. sanguinea has anti-mutagenic effect, but no mutagenicity. Cell culture in vitro experiments show that there is no inhibition of growth or increase in cell death on normal mouse fibroblasts, but a stronger inhibition of cell growth and an increase in cell death of Hep-2 and MGC-803 tumor cells. The results of this study illustrate that C. sanguinea extract has both anti-mutagenic and anti-tumor effects.     

Study of the heme catabolism of fish

• 1.1. The composition of bile pigments in the blood and bile of 39 species were studied.
• 2.2. Conjugated bilirubin (trace to 4.62 mg/100 ml) was detected in the serum of most fish, while biliverdin (trace to 2.0 mg/100ml) was detected only in Anguilla Japonica, Thalassoma lunare and Clinocottus analis.
• 3.3. Analysis showed tht there are two types of bile pigments excretion pattern in these fishes. The first pattern excretes bilirubin (most conjugate) predominantly, the other excretes mostly biliverdin with some bilirubin. However, during starvation, the excretion of conjugate bilirubin gradually shifted to unconjugated biliverdin. The rate of shifting varies with species.
• 4.4. Introduction of bilirubin into Anguilla japonica produced an initial excretion of mono-conjugates, followed by di-conjugates. Introduction of biliverdin caused an increased in the excretion of unconjugated biliverdin, but no significant increase of bilirubin in the bile was detected.
• 5.5. A binary excretion pathway of bile pigments in fish is proposed. The evolutionary characteristics of heme catabolism in terrestrial animals with respect to this pathway is discussed.

     

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).     

UDP-glucuronosyltransferase activity and bilirubin conjugation in the bullfrog.

Bile pigments of bile and serum of Rana catesbeiana were investigated by means of high-pressure liquid chromatography. The major pigment in both bile and serum was bilirubin IX alpha. Bilirubin UDP-glucuronosyltransferase activity was found in the livers of all animals examined, but no conjugated bilirubin was detectable in the bile. Frog bile was found to contain large amounts of beta-glucuronidase. When the beta-glucuronidase inhibitor saccharo-1,4-lactone was introduced into the gall bladder followed by an exogenous bilirubin load, bilirubin glucuronide appeared in the bile.     

Bilirubin binding activity of cytokeratin 18 isolated from the porcine liver

A porcine liver 40 kDa protein designated SBP40 isolated by affinity chromatography with agarose-linked spermine was identified as a porcine cytokeratin 18 on the basis of partial amino acid sequences of peptides derived by lysylendopeptidase digestion and by its reactivity with two commercially available preparations of monoclonal antibody. Immunohistochemistry revealed that SBP40 is localized at the hepatocyte membranes, preferentially in the bile canalicular area in accordance with the previously reported localization of cytokertain 18 in the murine liver. Affinity chromatography with agarose-linked bilirubin, a solubilization experiment of bilirubin from bilirubin-Sephadex G-10 complex, and gel-filtration of a mixture with bilirubin demonstrated that SBP40 or porcine cytokeratin 18 has binding affinity for bilirubin. These results suggest that cytokeratin 18 may play a role as a membrane reservoir in the event of transport and secretion of bile pigments in the liver.     

Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples.

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.     

Non-enzymic hydrolysis of bilirubin mono- and diglucuronide to unconjugated bilirubin in model and native bile systems. Potential role in the formation of gallstones.

Pigment gallstones contain considerable amounts of unconjugated bilirubin (UCB) in the form of calcium bilirubinate and/or bilirubin polymers. Since more than 98% of bile pigments are excreted as conjugates of bilirubin, the source of this UCB needs to be identified. By using a rapid h.p.l.c. method, we compared the non-enzymic hydrolysis of bilirubin monoglucuronide (BMG) and bilirubin diglucuronide (BDG) to UCB in model bile and in native guinea-pig bile. Model biles containing 50 microM solutions of pure BMG and BDG were individually incubated in 25 mM-sodium taurocholate (NaTC) and 0.4 M-imidazole/5 mM-ascorbate buffer (TC-BUF) at 37 degrees C. Over an 8 h period, BMG hydrolysis produced 4-6 times more UCB than BDG hydrolysis. At pH 7.4, 25% of the BMG was converted into UCB, whereas only 4.5% of BDG was converted into UCB. Hydrolysis rates for both BMG and BDG followed the pH order 7.8 greater than 7.6 approximately equal to 7.4 greater than 7.1 Incubation with Ca2+ (6.2 mM) at pH 7.4 in TC-BUF resulted in precipitated bile pigment which, at 100 X magnification, appeared similar to precipitates seen in the bile of patients with pigment gallstones. At pH 7.4, lecithin (crude phosphatidylcholine) (4.2 mM) was a potent inhibitor of hydrolysis of BMG and BDG. The addition of a concentration of cholesterol equimolar with that of lecithin eliminated this inhibitory effect. Guinea-pig gallbladder bile incubated with glucaro-1,4-lactone (an inhibitor of beta-glucuronidase) underwent hydrolysis similar to the model bile systems. The non-enzymic hydrolysis of bile pigments, especially BMG, may be an important mechanism of bile-pigment precipitation and, ultimately, of gallstone formation.