Toxicology, 75 (1992) 221-234 Elsevier Scientific Publishers Ireland l,td.
221
Individual serum bile acids as early indicators of carbon tetrachloride- and chloroform-induced liver injury* Cheng-long Bai a, Paul J. Canfield b and Neill H. Stacey a aToxicology Unit. National Institute ¢~f Occupational th'ahh and Sa./ety (Worksale Australia/ and ~Department of Veterinary Pathology, The l~"niver.~ity¢~/'Svdney. NSW ( AuMralia) (Received April 14th, 1992; accepted July 13th. 1992)
Summary Individual serum bile acids (SBA) are emerging as potentially useful early indicators of liver injury. This study was undertaken to compare the usefulness of individual SBA with the routinely used assays for detecting the effects of the hepatotoxicants carbon tetrachloride (CCI4) and chloroform (CHCI3). Serum samples were assayed for liver injury by determination of alanine aminotransferase (ALT), aspartate amino-transferase (AST), alkaline phosphatase (ALP), bilirubin and total bile acid (by enzymatic kit). These results were compared with levels of individual SBA measured by high performance liquid chromatography (HPLC). Liver samples from CCla-treated rats were taken for light and electron microscopic examination. The highest dose for each chemical caused increases in serum ALT and AST but not ALP. Chloroform at the highest dose increased bilirubin. Total SBA levels as assayed by the kit were elevated in response to CCI4 and CHCI 3 at doses below which serum enzymes and bilirubin were increased. Some individual SBA were increased at a still lower dose for each of these two chlorinated solvents. At the lowest dose of CCI4 tested no consistent light microscopic or ultrastructural changes were found. At all the higher doses periacinar cells displayed typical accumulation of lipid droplets and degranulation and dilation of rough cndoplasmic reticulum. The extent of the ultrastructural changes were dose-dependent. Thus individual SBA assayed by HPLC may be considered as a very sensitive indicator of liver injury induced by the classical hepatotoxicants carbon tctrachloride and chloroform.
Key word~." Serum bile acids; HPLC: Liver injury: Carbon tetrachloride (CCI4): Chloroform ((.;1t('13) Correspondence to." Neill H. Stacey, National Institute of Occupational Health and Safety, GPO Box 58, Sydney, NSW 2001, Australia. *This material was presented in part at the 13th Asian Conference on Occupational Health, Bangkok, Thailand, 1991. Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect those of the National Occupational Health and Safety Commission. Abbreviations." ALP, alkaline phosphatase: AI,I, alanine aminotransferase; AST. aspartate aminotransferase; BMC, 4-bromomethyl-7-methoxycoumarine; ('A, cholic acid: ("('14, carbon tetrachloride; CDC, chenodeoxycholic acid; CHCI 3, chloroform: DC, deoxycholic acid; G, Golgi complex: GC, glycocholic acid; GCDC, glycochenodeoxycholic acid: GDC, glycodeoxycholic acid; GLC, glycolithocholic acid; HPLC, high performance liquid chromatography; i.p., intraperitoneal injection; M, mitochondria; NCD, 23-nor-5/3-cholanic acid-3c~,12a-diaol: R ER, rough endoplasmic reticulum; SBA, serum bile acids; TC, taurocholic acid: TCDC. taurcx:henodcoxycholic Acid: TDC, taurodeoxycholic acid; TUDC, tauroursodeoxycholic acid; UDC, ursodeoxycholic acid. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
222 Introduction
It is documented that certain chemical agents used in industry, such as the chlorinated solvents carbon tetrachloride (CC14), chloroform (CHC13) and 1,1,2,2tetrachloroethane can cause serious hepatic injury. This list of potentially hepatotoxic chemicals is continually growing [1,2]. Concern about exposed workers has led to a demand for the development of laboratory tests to be used in the early diagnosis and prevention of occupational liver dysfunction [3,4]. Currently used indicators are, for instance, serum activities of 'liver enzymes' such as alanine aminotransferase (ALT), aspartate amino-transferase (AST) and alkaline phosphatase (ALP) and bilirubin. These tests have been used to screen for liver dysfunction in workers and experimental animals exposed to organic solvents. However, the limitations of standard biochemical enzyme studies in identifying the early phase or progression of liver injury have been recognised [5,6]. Serum enzyme studies are of limited value because they primarily reflect acute disruption of cell membrane integrity (liver cell leaking) rather than the uptake, metabolism, storage, or excretion function of the liver cells. Furthermore, some enzyme levels can also be increased in non-hepatic diseases [3]. There are reasons, therefore, to investigate the possible use of other more sensitive indicator and respective specific laboratory tests [3,7,8]. Recently, serum bile acids (SBA), have been measured in workers exposed to a variety of solvents [9-11]. Several techniques of SBA measurement, such as enzymatic tests, radioimmunoassay techniques, gas chromatography and high performance liquid chromatography (HPLC) were used [9,11-15]. It has been shown that exposure to the solvents is associated with elevations in total SBA levels when all other indicators are within the normal range. However, these studies have some limitations in that generally individual SBA were not assayed and exposure to a single individual solvent could not always be identified. Furthermore, there has been little subsequent comparative investigation in experimental animals [8]. The present experiments were, therefore, designed to investigate whether individual SBA would provide a more sensitive indicator of liver injury than the more routinely used tests. The chemicals selected for study are the well recognised hepatotoxicants CCI 4 and CHC13 [1,2,16-18]. Three doses on consecutive days were administered. Individual SBA were assayed with HPLC for comparison with results for established clinical indicators, such as ALT, AST, ALP and bilirubin. Total bile acid levels which are also used quite often were determined with an enzymatic kit for comparison to the data for the individual SBA. Histological examination, using light and transmission electron microscopy, was limited to normal and CCl4-affected livers, as the type of hepatotoxicity produced by CC14 is similar to that produced by CHCI 3. Materials and methods
Chemicals Carbon tetrachloride (CC14) and chloroform (CHC13), spectrosol grade, were obtained from Ajax Chemicals (Sydney, Australia). 4-Bromomethyl-7-methoxycoumarine (BMC), 18-crown-6 (1,4,7,10,13,16-hexaoxacylooctadecane), cholyl-
223 glycine hydrolase (42 units/mg protein) and all bile acid standards: sodium salt of cholic acid (CA), ursodeoxycholic acid (UDC), deoxycholic acid (DC), chenodeoxycholic acid (CDC), glycocholic acid (GC), glycodeoxycholic acid (GDC), glycochenodeoxycholic acid (GCDC), glycolithocholic acid (GLC), taurocholic acid (TC), taurochenodeoxycholic acid (TCDC), tauroursodeoxycholic acid (TUDC) and taurodeoxycholic acid (TDC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile and methanol, HPLC grade, were supplied by Mallinckrodt Australia Pry. Ltd. (Melbourne, Australia). 23-Nor-5/3-cholanic acid-3a,12a-diaol (also called 3~-12~-dihydroxynor-cholanate; nordeoxycholic acid) (NCD), TLC one spot purity, was purchased from Steraloids Inc. (Wilton, NH, USA) as an internal standard for HPLC assay. Animals and treatment
Male Sprague-Dawley rats from The University of Sydney animal house weighing 250-300 g were used. They were allowed free access to food (Allied Stock Feeds, Sydney, Australia) and water. CCi4 (0.1, 0.5, 1.0 and 5.0 mmol/kg body wt.) and CHCI3 (0.1, 0.5, 1.0, 5.0 and 7.5 mmol/kg body wt.), dissolved in corn oil (1 ml/kg body wt.), were administered via intraperitoneal (i.p.) injection to rats on each of 3 consecutive days at about 09:00 h. Doses were selected on the basis of literature values and preliminary experiments. All controls were given 1 ml/kg body wt. of corn oil in the same manner. Unless otherwise stated there were 4 animals per group. Food was removed from animals after the last treatment and 4 h later blood samples were taken from the abdominal aorta under ether anesthesia at about 13:00-14:00 h for each group. The sera from the blood samples were stored at -20°C until assayed within the next few days. Serum enzyme and bilirubin assays
ALT, AST and ALP were assayed with enzymatic methods using a Roche CentrifiChem (Model 400) autobiochemical analyser and the appropriate kits - S.V.R. TM ALT test kit and S.V.R. TM AST test kit, Behring Diagnostic Inc. (Somerville, NJ, USA); ALP (ROCHE) Uni-kit II and bilirubin (ROCHE) Uni-kit II from Hoffman-La Roche Diagnostica (Basle, Switzerland). Total serum bile acid
Total serum bile acids were measured with a PYE Unicam PU8800 UV/VIS spectrophotometer using an Enzabile kit supplied by NYCOMED AS. (Oslo, Norway). Individual serum bile acids assay
The method for assay of individual bile acids in serum followed that of Wang et al. [19] with some modifications. A Waters HPLC system with Maxima-820 chromatography workstation (Waters Chromatography Division, Milford, MA, USA) (Model 501 pumps, Model 715 Ultra WISP sample processor, blue chip CM-6260 personal computer, Nova-Pak C18 column and Model 470 scanning fluorescence detector) were used. Serum samples spiked with internal standard (NCD) were mixed with cold acetone for deproteinization then prepared with a
224 Sep-Pak C 18 cartridge. The methanol eluate was dried down at 60°C with a JOUAN vacuum centrifugal evaporator, RC 1010 (Saint-Herblain, France). HPLC was run with two mobile phases (containing with methanol, acetonitrile and water, 15:14:71) under a convex gradient. Detector excitation and emission wavelengths were 320 and 385 nm, respectively.
Histology The left lateral lobe of the liver was removed immediately after blood sampling and sliced. Tissues for light microscopy were fixed in neutral-buffered formalin. These were processed and stained with hematoxylin and eosin (H&E) by standard techniques. Liver slices for ultrastructural examination were immersed in 2.5% glutaraldehyde in 0.1 M phosphate buffer then cut into cubes measuring less than 1 mm 3. These were fixed in 2.5% glutaraldehyde on a mixing rotator for 1 h and then post-fixed in 1% phosphate-buffered osmium tetroxide for 1 h. The specimens were dehydrated through graded alcohol and acetone solutions and embedded in Spurr's resin (65°C, overnight). Specimens were sectioned on an Ultracut E Ultramicrotome (ReichertJung Optische Werke AG, Vienna, Austria) and double stained with uranyl acetate in 30% ethanol and lead citrate prior to examination with a JEOL-JEM-100CX Transmission Electron Microscope (JEO-JEM, Tokyo, Japan).
Data analysis Results were statistically evaluated by an analysis of variance and Duncan's test [20]. Significance level was set at P < 0.05. Results
The release of ALT and AST on exposure to the highest dose of CC14 (5.0 mmol/kg) or CHC13 (7.5 mmol/kg) is shown in Fig. 1. The lower doses did not produce any increase in ALT and AST, while ALP was not affected at any dose of these solvents. For bilirubin, the only significant elevation was found with the highest dose (7.5 mmol/kg) of CHCI3 (Table I). In the assay of sera from CC14- or CHC13-dosed rats for total bile acids with the Enzabile kit, a marked effect of exposure was found, being evident at doses as low as 1 mmol/kg (Fig. 2). The results for individual SBA levels determined by HPLC assay are shown in Tables II and III for CC14 and CHCI3, respectively. The mean levels of totalled SBA with HPLC assay were increased in the exposed groups, with significant differences occurring down to the next to lowest dose of CC14 or CHCI 3 (0.5 mmol/kg). As shown in Figs. 3 and 4 mean levels of three groups of subtotalled SBA, free, glycine and taurine, were increased in the rats. In CC14-exposed rats, the lowest dose (0.1 mmol/kg) caused increases in free and glycine-conjugated bile acids, while taurine-conjugated bile acid levels were increased from 0.5 mmol/kg dose. In CHCl3-treated rats, the taurine conjugates were increased from the 0.5 mmol/kg dose, the free from 1.0 mmol/kg dose while the glycine conjugates were only elevated at the highest dose.
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221
Individual serum bile acids as early indicators of carbon tetrachloride- and chloroform-induced liver injury* Cheng-long Bai a, Paul J. Canfield b and Neill H. Stacey a aToxicology Unit. National Institute ¢~f Occupational th'ahh and Sa./ety (Worksale Australia/ and ~Department of Veterinary Pathology, The l~"niver.~ity¢~/'Svdney. NSW ( AuMralia) (Received April 14th, 1992; accepted July 13th. 1992)
Summary Individual serum bile acids (SBA) are emerging as potentially useful early indicators of liver injury. This study was undertaken to compare the usefulness of individual SBA with the routinely used assays for detecting the effects of the hepatotoxicants carbon tetrachloride (CCI4) and chloroform (CHCI3). Serum samples were assayed for liver injury by determination of alanine aminotransferase (ALT), aspartate amino-transferase (AST), alkaline phosphatase (ALP), bilirubin and total bile acid (by enzymatic kit). These results were compared with levels of individual SBA measured by high performance liquid chromatography (HPLC). Liver samples from CCla-treated rats were taken for light and electron microscopic examination. The highest dose for each chemical caused increases in serum ALT and AST but not ALP. Chloroform at the highest dose increased bilirubin. Total SBA levels as assayed by the kit were elevated in response to CCI4 and CHCI 3 at doses below which serum enzymes and bilirubin were increased. Some individual SBA were increased at a still lower dose for each of these two chlorinated solvents. At the lowest dose of CCI4 tested no consistent light microscopic or ultrastructural changes were found. At all the higher doses periacinar cells displayed typical accumulation of lipid droplets and degranulation and dilation of rough cndoplasmic reticulum. The extent of the ultrastructural changes were dose-dependent. Thus individual SBA assayed by HPLC may be considered as a very sensitive indicator of liver injury induced by the classical hepatotoxicants carbon tctrachloride and chloroform.
Key word~." Serum bile acids; HPLC: Liver injury: Carbon tetrachloride (CCI4): Chloroform ((.;1t('13) Correspondence to." Neill H. Stacey, National Institute of Occupational Health and Safety, GPO Box 58, Sydney, NSW 2001, Australia. *This material was presented in part at the 13th Asian Conference on Occupational Health, Bangkok, Thailand, 1991. Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect those of the National Occupational Health and Safety Commission. Abbreviations." ALP, alkaline phosphatase: AI,I, alanine aminotransferase; AST. aspartate aminotransferase; BMC, 4-bromomethyl-7-methoxycoumarine; ('A, cholic acid: ("('14, carbon tetrachloride; CDC, chenodeoxycholic acid; CHCI 3, chloroform: DC, deoxycholic acid; G, Golgi complex: GC, glycocholic acid; GCDC, glycochenodeoxycholic acid: GDC, glycodeoxycholic acid; GLC, glycolithocholic acid; HPLC, high performance liquid chromatography; i.p., intraperitoneal injection; M, mitochondria; NCD, 23-nor-5/3-cholanic acid-3c~,12a-diaol: R ER, rough endoplasmic reticulum; SBA, serum bile acids; TC, taurocholic acid: TCDC. taurcx:henodcoxycholic Acid: TDC, taurodeoxycholic acid; TUDC, tauroursodeoxycholic acid; UDC, ursodeoxycholic acid. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
222 Introduction
It is documented that certain chemical agents used in industry, such as the chlorinated solvents carbon tetrachloride (CC14), chloroform (CHC13) and 1,1,2,2tetrachloroethane can cause serious hepatic injury. This list of potentially hepatotoxic chemicals is continually growing [1,2]. Concern about exposed workers has led to a demand for the development of laboratory tests to be used in the early diagnosis and prevention of occupational liver dysfunction [3,4]. Currently used indicators are, for instance, serum activities of 'liver enzymes' such as alanine aminotransferase (ALT), aspartate amino-transferase (AST) and alkaline phosphatase (ALP) and bilirubin. These tests have been used to screen for liver dysfunction in workers and experimental animals exposed to organic solvents. However, the limitations of standard biochemical enzyme studies in identifying the early phase or progression of liver injury have been recognised [5,6]. Serum enzyme studies are of limited value because they primarily reflect acute disruption of cell membrane integrity (liver cell leaking) rather than the uptake, metabolism, storage, or excretion function of the liver cells. Furthermore, some enzyme levels can also be increased in non-hepatic diseases [3]. There are reasons, therefore, to investigate the possible use of other more sensitive indicator and respective specific laboratory tests [3,7,8]. Recently, serum bile acids (SBA), have been measured in workers exposed to a variety of solvents [9-11]. Several techniques of SBA measurement, such as enzymatic tests, radioimmunoassay techniques, gas chromatography and high performance liquid chromatography (HPLC) were used [9,11-15]. It has been shown that exposure to the solvents is associated with elevations in total SBA levels when all other indicators are within the normal range. However, these studies have some limitations in that generally individual SBA were not assayed and exposure to a single individual solvent could not always be identified. Furthermore, there has been little subsequent comparative investigation in experimental animals [8]. The present experiments were, therefore, designed to investigate whether individual SBA would provide a more sensitive indicator of liver injury than the more routinely used tests. The chemicals selected for study are the well recognised hepatotoxicants CCI 4 and CHC13 [1,2,16-18]. Three doses on consecutive days were administered. Individual SBA were assayed with HPLC for comparison with results for established clinical indicators, such as ALT, AST, ALP and bilirubin. Total bile acid levels which are also used quite often were determined with an enzymatic kit for comparison to the data for the individual SBA. Histological examination, using light and transmission electron microscopy, was limited to normal and CCl4-affected livers, as the type of hepatotoxicity produced by CC14 is similar to that produced by CHCI 3. Materials and methods
Chemicals Carbon tetrachloride (CC14) and chloroform (CHC13), spectrosol grade, were obtained from Ajax Chemicals (Sydney, Australia). 4-Bromomethyl-7-methoxycoumarine (BMC), 18-crown-6 (1,4,7,10,13,16-hexaoxacylooctadecane), cholyl-
223 glycine hydrolase (42 units/mg protein) and all bile acid standards: sodium salt of cholic acid (CA), ursodeoxycholic acid (UDC), deoxycholic acid (DC), chenodeoxycholic acid (CDC), glycocholic acid (GC), glycodeoxycholic acid (GDC), glycochenodeoxycholic acid (GCDC), glycolithocholic acid (GLC), taurocholic acid (TC), taurochenodeoxycholic acid (TCDC), tauroursodeoxycholic acid (TUDC) and taurodeoxycholic acid (TDC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile and methanol, HPLC grade, were supplied by Mallinckrodt Australia Pry. Ltd. (Melbourne, Australia). 23-Nor-5/3-cholanic acid-3a,12a-diaol (also called 3~-12~-dihydroxynor-cholanate; nordeoxycholic acid) (NCD), TLC one spot purity, was purchased from Steraloids Inc. (Wilton, NH, USA) as an internal standard for HPLC assay. Animals and treatment
Male Sprague-Dawley rats from The University of Sydney animal house weighing 250-300 g were used. They were allowed free access to food (Allied Stock Feeds, Sydney, Australia) and water. CCi4 (0.1, 0.5, 1.0 and 5.0 mmol/kg body wt.) and CHCI3 (0.1, 0.5, 1.0, 5.0 and 7.5 mmol/kg body wt.), dissolved in corn oil (1 ml/kg body wt.), were administered via intraperitoneal (i.p.) injection to rats on each of 3 consecutive days at about 09:00 h. Doses were selected on the basis of literature values and preliminary experiments. All controls were given 1 ml/kg body wt. of corn oil in the same manner. Unless otherwise stated there were 4 animals per group. Food was removed from animals after the last treatment and 4 h later blood samples were taken from the abdominal aorta under ether anesthesia at about 13:00-14:00 h for each group. The sera from the blood samples were stored at -20°C until assayed within the next few days. Serum enzyme and bilirubin assays
ALT, AST and ALP were assayed with enzymatic methods using a Roche CentrifiChem (Model 400) autobiochemical analyser and the appropriate kits - S.V.R. TM ALT test kit and S.V.R. TM AST test kit, Behring Diagnostic Inc. (Somerville, NJ, USA); ALP (ROCHE) Uni-kit II and bilirubin (ROCHE) Uni-kit II from Hoffman-La Roche Diagnostica (Basle, Switzerland). Total serum bile acid
Total serum bile acids were measured with a PYE Unicam PU8800 UV/VIS spectrophotometer using an Enzabile kit supplied by NYCOMED AS. (Oslo, Norway). Individual serum bile acids assay
The method for assay of individual bile acids in serum followed that of Wang et al. [19] with some modifications. A Waters HPLC system with Maxima-820 chromatography workstation (Waters Chromatography Division, Milford, MA, USA) (Model 501 pumps, Model 715 Ultra WISP sample processor, blue chip CM-6260 personal computer, Nova-Pak C18 column and Model 470 scanning fluorescence detector) were used. Serum samples spiked with internal standard (NCD) were mixed with cold acetone for deproteinization then prepared with a
224 Sep-Pak C 18 cartridge. The methanol eluate was dried down at 60°C with a JOUAN vacuum centrifugal evaporator, RC 1010 (Saint-Herblain, France). HPLC was run with two mobile phases (containing with methanol, acetonitrile and water, 15:14:71) under a convex gradient. Detector excitation and emission wavelengths were 320 and 385 nm, respectively.
Histology The left lateral lobe of the liver was removed immediately after blood sampling and sliced. Tissues for light microscopy were fixed in neutral-buffered formalin. These were processed and stained with hematoxylin and eosin (H&E) by standard techniques. Liver slices for ultrastructural examination were immersed in 2.5% glutaraldehyde in 0.1 M phosphate buffer then cut into cubes measuring less than 1 mm 3. These were fixed in 2.5% glutaraldehyde on a mixing rotator for 1 h and then post-fixed in 1% phosphate-buffered osmium tetroxide for 1 h. The specimens were dehydrated through graded alcohol and acetone solutions and embedded in Spurr's resin (65°C, overnight). Specimens were sectioned on an Ultracut E Ultramicrotome (ReichertJung Optische Werke AG, Vienna, Austria) and double stained with uranyl acetate in 30% ethanol and lead citrate prior to examination with a JEOL-JEM-100CX Transmission Electron Microscope (JEO-JEM, Tokyo, Japan).
Data analysis Results were statistically evaluated by an analysis of variance and Duncan's test [20]. Significance level was set at P < 0.05. Results
The release of ALT and AST on exposure to the highest dose of CC14 (5.0 mmol/kg) or CHC13 (7.5 mmol/kg) is shown in Fig. 1. The lower doses did not produce any increase in ALT and AST, while ALP was not affected at any dose of these solvents. For bilirubin, the only significant elevation was found with the highest dose (7.5 mmol/kg) of CHCI3 (Table I). In the assay of sera from CC14- or CHC13-dosed rats for total bile acids with the Enzabile kit, a marked effect of exposure was found, being evident at doses as low as 1 mmol/kg (Fig. 2). The results for individual SBA levels determined by HPLC assay are shown in Tables II and III for CC14 and CHCI3, respectively. The mean levels of totalled SBA with HPLC assay were increased in the exposed groups, with significant differences occurring down to the next to lowest dose of CC14 or CHCI 3 (0.5 mmol/kg). As shown in Figs. 3 and 4 mean levels of three groups of subtotalled SBA, free, glycine and taurine, were increased in the rats. In CC14-exposed rats, the lowest dose (0.1 mmol/kg) caused increases in free and glycine-conjugated bile acids, while taurine-conjugated bile acid levels were increased from 0.5 mmol/kg dose. In CHCl3-treated rats, the taurine conjugates were increased from the 0.5 mmol/kg dose, the free from 1.0 mmol/kg dose while the glycine conjugates were only elevated at the highest dose.
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