Chem. Res. Toxicol. 1994, 7, 850-856
850
Hepatotoxicity of Germander (Teucrium chamaedrys L.) and One of Its Constituent Neoclerodane Diterpenes Teucrin A in the Mouse Samir A. Kouzi,t>$Randolph J. McMurtry,S and Sidney D. Nelson*>? Department of Medicinal Chemistry, BG-20, School of Pharmacy, University of Washington, Seattle, Washington 98195, and Department of Pathology, Presbyterian 1st. Luke's Denver Hospital, Denver, Colorado 80218 Received July 18, 1994@
The hepatotoxicity of the herbal plant germander and that of one of its major furanoneoclerodane diterpenes, teucrin A, were investigated in mice. Teucrin A was found t o cause the same midzonal hepatic necrosis a s observed with extracts of the powdered plant material. Evidence that bioactivation of teucrin A by cytochromes P450 (P450) to a reactive metaboliteb) is required for initiation of the hepatocellular damage is provided by results of experiments on the induction and inhibition of P450 and from studies on the effects of glutathione depletion. Pretreatment of mice with the P450 inducer phenobarbital enhanced the hepatotoxic response, a s indicated by a n increase in plasma alanine aminotransferase (ALT) levels and hepatic necrosis, while pretreatment with the P450 inhibitor piperonyl butoxide markedly attenuated the toxic response. Hepatotoxicity of teucrin A also was increased following pretreatment with the inhibitor of glutathione synthesis buthionine sulfoximine. Most importantly, the tetrahydrofuran analog of teucrin A, obtained by selective chemical reduction of the furan ring, was not hepatotoxic, a result that provides strong evidence that oxidation of the furan ring moiety of the neoclerodane diterpenes is involved in the initiation of hepatocellular injury caused by germander.
Introduction Wild germander (Teucrium chamaedrys L.) is an herbal plant of the Labiatae family that has been used as a folk medicine for several purposes, ranging from the treatment of rheumatism to use as a diuretic and antiseptic agent (1, 2). In 1991, capsules containing either germander powder alone, or in combination with camellia tea, were marketed in France for use in weight control, and 30 cases of heptatotoxicity have been reported to result from the use of these products (3-6) including a fatality due to fulminant hepatic necrosis (7). As a consequence, preparations containing germander were prohibited in France. Earlier this year, Loeper et al. (8) reported results of studies in mice which implicate cytochrome P450 (P450)' metabolism of the neoclerodane diterpenoids, present in aerial parts of T. chamaedrys, in hepatotoxicity caused by germander. We also have been investigating the mechanism of hepatotoxicity caused by this plant based on the fact that the neoclerodanes are furan-containing diterpenoids (9, lo), and based on the results of our previous studies (11, 12) of the terpenoid menthofuran * To whom correspondence should be addressed at the Department of Medicinal Chemistry, BG-20, University of Washington, Seattle, WA
98195. + University of Washington. Present address: Department of Medicinal Chemistry, School of Pharmacv, Northeast Louisiana University, Monroe, LA 71209. 8 PresbyteriadSt. Luke's Denver Hospital. EI Abstract published in Advance ACS Abstracts, October 15, 1994. Abbreviations: GSH, reduced glutathione; BSO, L-buthionine (S,R)-sulfoximine; ALT, alanine aminotransferase; SFU, SigmaFrankel units; PBO, piperonyl butoxide; DTNB, 5,5'-dithiobis(2nitrobenzoic acid); PB, sodium phenobarbital; FAB/MS, fast atom bombardment mass spectrometry; 2-HEDS, 2-hydroxyethyl disulfide; COSY, homonuclear correlation spectroscopy; APT, attached proton test; P450, cytochrome P450.
*
as a hepatotoxic metabolite of the monoterpene, OW+)pulegone, a major constituent of pennyroyal oil, another herbal folklore medicine that has caused severe hepatotoxicity and deaths in humans (13, 14). Results of our studies on germander corroborate those of Loeper et al. (8) and extend them to show that the furan ring of one of the major neoclerodane diterpenoids of germander, teucrin A (15), is required for it to cause hepatotoxicity in mice.
Experimental Procedures General. Melting points were determined on a ThomasHoover capillary melting point apparatus in open capillary tubes and are uncorrected. Optical rotations were measured with a Jasco DIP-4 digital polarimeter in pyridine or methanol. IR spectra were recorded in KBr using a Perkin-Elmer 1600 FTIR spectrophotometer. The term in vacuo refers to removal of solvent with a rotary evaporator under water aspirator vacuum (15-30 mmHg). 'H- and 13C-NMR spectra were obtained in pyridine-ds on a Varian VXR-300 FT spectrometer operating at 300 and 75 MHz, respectively. Standard Varian pulse sequences were used for homonuclear correlation spectroscopy (COSY) and attached proton test (APT) experiments. The chemical shift values are reported in ppm, and the coupling constants ( J values) are in Hz. Electron impact mass spectra were obtained using a VG7070H mass spectrometer (VG Analytical, Manchester, England) with ion source temperature at 200 "C, emission current at 100 PA, and accelerating voltage of 4 kV. Spectra were recorded at a nominal resolution of MIAM = 1000 (10% valley). Magnetic field scanning from m / z 50 to 400 was repeated at 1.5 ddecade. Fast atom bombarbment mass spectrometry (FABMS) spectra were recorded in 2-hydroxyethyl disulfide (2-HEDS) matrix on a VG 70-SEQ hybrid tandem mass spectrometer equipped with an Ion Tech saddlefield fast atom gun and a VG 11/250 data system. Ionization was achieved following bombardment with a primary beam of xenon (8 KeV), and spectra were recorded at an accelerating voltage of 8 kV and a nominal mass resolution of M/AM = 2000
0 1994 American Chemical Society
Hepatotoxicity of Germander a n d Teucrin A (10% valley). High resolution (M/AM = 10 000) FAB/MS spectra were recorded utilizing thioglycerol as matrix and PEG 300 as reference. TLC analyses were carried out on precoated silica gel 60 F254 plates (E. Merck, Darmstadt). The adsorbent used for column chromatography was silica gel 60/70-230 mesh (Aldrich Chemical Co., Milwaukee, WI). The developing system used for TLC was methanolkhloroform (4:96 v/v), and visualization of the TLC plates was performed using both anisaldehyde/ sulfuric acid spray reagent and W light (254 nm). The spots were visualized by spraying the plate with the spray reagent and then heating it a t 110 "C for 5 min in an oven. Chemicals. Sodium phenobarbital (PB) was obtained from Spectrum Chemical Co. (Gardena, CA) and piperonyl butoxide (PBO) from Matheson, Coleman and Bell (Norwood, OH). A small amount of authentic teucrin A was obtained from Professor Benjamin Rodriguez (CSIC, Instituto De Quimica Organica General, Madrid, Spain). Rhodium on carbon (5%)was obtained from Aldrich Chemical Co. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Solvents were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). HPLC Analyses. Analysis of pure teucrin A as well as teucrin A in the acetone extract of germander by HPLC was carried out on a C-18, 5-pm Adsorbosphere column (250 mm x 4.6 mm, Alltech, Deerfield, IL) coupled to a 10 mm x 4.6 mm guard column utilizing a H P 1090 Series I1 liquid chromatograph equipped with diode-array UV/vis detector. The injection volume was 1pL, and the flow rate was 1 m u m i n . The eluent was monitored a t I = 220 nm, and the following gradient system was utilized: 0 min, 10% MeOWH20; 10 min, 70%MeOWH20; 15 min, 70% MeOWHzO; 20 min, 10% MeOWH20; 25 min, 10% MeOWHzO. For the diastereomeric separation of tetrahydroteucrin A the following gradient system was utilized: 0 min, 10% acetonitrile/water; 2 min, 10% acetonitrile/water; 62 min, 15% acetonitrile/water; 122 min, 15% acetonitrile/water; 125 min, 95% acetonitrile/water; 130 min, 95% acetonitrile/water; 133 min, 10% acetonitrile/water. Plant Collection and Identification. The aerial parts of germander (Teucrium chamaedrys L.,family Labiatae) were collected from the University of Washington campus in Seattle, WA on September 8, 1992. The plant was identified by Dr. Melinda F. Denton, Professor of Botany and Curator of the Herbarium, Department of Botany, University of Washington, and Mr. Douglas M. Ewing, Greenhouse Manager, Department of Botany, University of Washington. A voucher specimen was prepared and deposited a t the Herbarium, Department of Botany, University of Washington. The plant material was then dried a t 30 "C in an oven and ground in a Wiley Mill to yield 3 kg of finely powdered germander. This material will be referred to a s "Washington germander". A commercial source of germander was subsequently located, and the plant was purchased as finely powdered material from Dixa AG Co. for Medicinal Herbs and Spices (St. Gallen, Switzerland). This material will be referred to as "European germander". A literature survey of ethnomedical information on germander, as well as of the phytochemistry and biological activities for extracts of germander, was obtained by searching the NAPRALERT database, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois a t Chicago, Chicago, IL 60612.
Preparation of Crude Ethanol and Acetone Extracts of Germander and Isolation of Teucrin A. Powdered Washington germander (1 kg) was percolated a t room temperature with 95% ethanovwater (13 L) for 15 days. Evaporation of ethanol in Vacuo a t 30 "C yielded 165 g of a crude gummy residue. Percolation of 1kg of powdered Washington germander with acetone (10 L) a t room temperature for 10 days followed by evaporation of solvent in uacuo a t 30 "C yielded 70 g of a crude acetone extract. A similar extract was prepared from the European germander by percolating 400 g of powdered germander in acetone (6 L) a t room temperature for 9 days. Evaporation of solvent in uacuo a t 30 "C yielded 45 g of acetone extract.
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 851 Column chromatography of 45 g of the acetone extract of the European germander over silica gel (1.4 kg) using methanol/ chloroform as eluent, followed by crystallization from acetone, yielded 460 mg of teucrin A as white needles (0.115% of the dry weight). Similarly, a total of 41 g of the acetone extract of Washington germander was chromatographed over silica gel to yield 300 mg of teucrin A (0.03% of the dry weight). Isolated teucrin A was identical in all respects (mp, mixed mp, IR, W, TLC, optical rotation, NMR, HPLC, EIMS) to an authentic sample of teucrin A and t o the reported literature data on teucrin A (9, 15, 16). Synthesis of Tetrahydroteucrin A. Teucrin A (60 mg, 0.174 mmol) was hydrogenated over 5%rhodium on carbon (70 mg) in glacial acetic acid (5 mL). The reaction mixture was first stirred under high vacuum for 15 min and then under hydrogen gas a t 26 W 7 6 0 mmHg for 10 h. The reaction mixture was filtered, and the acetic acid solution was evaporated to dryness under high vacuum. Column chromatography of the residue over silica gel utilizing MeOWCHC13 (5:95) as solvent, followed by recrystallization from acetone, yielded tetrahydroteucrin A a s white crystals (25 mg, 42% yield). HPLC analysis revealed that the product was '95% pure and that it was a mixture (44: 56) of two epimers a t carbon-13 with the following physical and spectral data: Rf 0.346 (MeOWCHC13, 10:90; teucrin A, Rf 0.474); mp 200-204 "C; [ a ] 2 8 = 0 c$145" ~ (c = 0.1, MeOH); IR (KBr) umax (cm-l): 3372, 2928, 1752, 1726, 1364, 1207, 1024, 965, 758, 595; W (acetonitrile): Amax = 218 ( E 1690); FABMS (positive ion) m / z 349 [MHl+,371 [M Nal+; high-resolution FABMS: 349.1641 (C1QHz506, [MHlf, calcd 349.1651); lH-NMR (pyridine-ds): 6 5.25 (m, l H , Hs), 4.99 (br d, l H , J = 2.7 Hz, exchangeable with DzO, OH), 4.59 (m, l H , Hlz), 4.29 (m, l H , HT),3.87 (m, 2H, H15 and H16), 3.70 (m, 2H, HI5 and H d , 2.79 (m, l H , Hlo), 2.42 (m, 3H, H11 and HIS), 2.24-1.30 (m, 9H, HI, Hz, H3, HE, and 1.26 (two d, 3H, J = 7.2 Hz, CH3); 13CNMR (pyridine-ds): 6 22.6 (C-l),25.5 (C-2),20.7(C-31, 128.8 (C4), 160.2 (C-5), 82.1 ((2-61, 73.3 (C-71, 39.3 (C-81, 57.6 and 57.7 (C-91, 42.8 (C-lo), 40.8 and 41.1 ( C - l l ) , 82.8 and 83.3 (C-12), 45.7 and 46.2 ((2-131, 28.5 and 29.6 (C-141, 68.6 and 68.9 (CE),69.5 and 71.2 (C-161, 14.7 (C-17), 174.7 (C-181, 182.1 (C19). Animals and Treatments. Male Swiss-Webster albino mice weighing 20-25 g (Charles River Laboratories, Wilmington, MA) were utilized in this study. Animals had free access to food (Animal Specialties, Hubbard, OR) and water and were housed in a temperature-controlled (72 & 4 "C) facility with a 12 h darkflight cycle for a t least 5 days after receipt and before treatment. At least 20 h before dosing, animals were denied access to food. All samples were administered to mice by oral gavage using a 18 gauge x 4 cm gavage needle with rounded tip. The vehicle used for all treatments (0.5 mL) was Tween 8O/corn oilhaline (2:2:6). For P450-induction experiments, PB was added to the animals' drinking water (0.1%) for 5 days prior to treatment. For P45O-inhibition experiments, PBO was given ip (1.36 g k g ) as neat liquid 30 min before treatment. For GSH-depletion experiments, L-buthionine (S,R)-sulfoximine (BSO) was administered ip (6 mmolkg) as a solution in normal saline 5 h before treatment. Assessment of Hepatotoxicity. Mice were decapitated and exsanguinated by collecting blood in heparinized beakers 24 h postdosing. Sections of liver were k e d in buffered formalin and sent to the Pathology Department, PresbyteriadSt. Luke's Hospital (Denver, CO) for the preparation of paraffin sections and staining with hematoxylin and eosin. The severity of hepatic necrosis in blinded samples was quantified as previously described (17): 0, absent; 1+, necrosis of less than 6% of hepatocytes; 2+, necrosis of 6-25% of hepatocytes; 3+, 26-50% of hepatocytes; and 4+, greater than 50% of hepatocytes. Blood samples were collected 24 h following treatments except during the time-course study of GSH levels, where blood samples were collected a t 0, 1, 3, 6, 12, and 24 h following treatment. Plasma alanine aminotransferase (ALT) levels in mice were determined as follows: Mice were decapitated and
+
852 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
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15
CH3
i Teucrln A
Tetrahydroteucrin A
il
Figure 1. Chemical structures of teucrin A and tetrahydroteucrin A.
exsanguinated by collecting blood in heparinized beakers. Blood samples were then centrifuged at 16000g for 8 min at room temperature, and the plasma was separated and stored immediately in the refrigerator at 4 "C. ALT levels were determined in plasma samples utilizing the commercially available Sigma Kit (Sigma Chemical Co.) for the quantitative, colorimetric determination of ALT in biological fluids. For liver GSH levels, mice were killed by decapitation and livers were excised immediately and kept on ice and were used within 15 min. Hepatic GSH was determined as total soluble thiols according t o the method of Ellman (18). Livers were minced and homogenized in 4 volumes of ice-cold 0.1 M phosphate buffer (pH 7.4). Protein was precipitated by addition of 1mL of 4% 5-sulfosalicylic acid and centrifuged at lOOOg for 15 min at 4 "C. A n aliquot (0.5 mL) of the supernatant was diluted to 5 mL with 0.1 M phosphate buffer (pH 8.0) before adding 50 pL of 5,5'-dithiobis(Z-nitrobenzoicacid) (DTNB)(4mg/ mL) and vortexed. Absorbance at 412 nm was measured 2030 s aRer addition of (DTNB).Liver GSH levels were determined 24 h following treatment for the GSH-depletionexperiment and at 0, 1, 3,6,12,and 24 h following treatment for the timecourse study of liver GSH levels.
Results Isolation and Identification of Teucrin A. Teucrin A (Figure 1)is one of the major neoclerodane diterpenoids present in germander (9). Therefore, we isolated it from an acetone extract of germander by column chromatography and crystallization and quantitated it by HPLC in acetone extracts in germander obtained on campus at the University of Washington and from a European commercial source. A standard curve, based on peak areas exhibited by various concentrations of pure teucrin A ( t =~10.4 min), was linear (r2= 0.997) over a range of 0.05-2.0 mg/mL. Teucrin A concentrations in the acetone extract of the Washington germander were 1.09% (0.04% of dry weight) and in the European germander were 1.58% (0.17% of dry weight). Synthesis and Characterization of Tetrahydroteucrin A. In order to assess the role of the furan ring in the hepatotoxicity of teucrin A, we synthesized the corresponding tetrahydrofuran analog, tetrahydroteucrin A (Figure 1). Mild catalytic hydrogenation of teucrin A with a rhodium on carbon catalyst yielded the desired compound without opening or reduction of any of the lactone rings as determined by the IR data and the molecular weight determined by FAB/MS. This was confirmed by lH- and 13C-NMRassignments (Experimental Procedures) which were based on 1D- and 2D-NMR experiments as well as comparisons of the chemical shift
-
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.
1
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1
o V
v "
1 "
o
v
1
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o
- m
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Figure 2. COSY plot of tetrahydroteucrin A. Standard Varian pulse sequence was utilized with a relaxation delay of 1.0 s and 90"pulse width. The data was pseudo-echo processed.
values and multiplicities with those of teucrin A (15,16). Most notably, the 13C spectrum shows absorbances for the two lactone carbonyls at 174.7 ppm (C-18) and 182.1 ppm (C-191, and the APT showed that the only differences in structure between teucrin A and tetrahydroteucrin A occurred in the furan ring. Finally, a COSY pulse sequence was used for individual 'H-NMR assignments (Figure 2). The chemical shifts and coupling patterns for all protons, except those at C-12 and the furan ring, are very similar to those reported for teucrin A (15). The furanyl aromatic protons are lost in tetrahydroteucrin A with the appearance of two distinct new sets of signals a t 3.70 and 3.87 ppm for the hydrogens on the ether carbons C-15 and (2-16. In addition, the signal for the hydrogen on C-12, the carbon connected to the tetrahydrofuran ring, at 4.59 ppm is shifted approximately 1ppm upfield from its position in teucrin A (5.75 ppm) consistent with the loss of aromaticity in the furan ring. More importantly, this proton is coupled to a new proton at C-13 as seen in the COSY plot (Figure 2). The complexity of the proton signal at C-12, and the fact that the methyl group at C-17 resonated as two doublets at 1.26 ppm, suggests that C-13 may be an epimeric center. Epimers would be anticipated from the reduction and are consistent with the 13C-NMRdata where doubling of many 13C signals (see experimental) was observed. This was confirmed by HPLC analysis of a sample of tetrahydroteucrin A, which showed two peaks with identical UV spectra. The area ratio of the peaks was 4456. Hepatotoxicity Studies. Administration of ethanolic extract (2-4 g) of Washington germander to mice caused hepatocellular injury in mice based on both large increases in plasma ALT concentrations and histopathological evidence of midzonal hepatic necrosis 24 h aRer dosing (Table 1). Administration of the acetone extract (0.5 gkg) from the same source caused similar effects that were markedly attenuated by pretreatment with the
Chem. Res. Toxicol., Vol. 7, No. 6,1994 853
Hepatotoxicity of Germander and Teucrin A Table 1. Plasma ALT Concentrations and Hepatic Necrosis Scores in Swiss-Webster Mice after the Administration of Extracts of Washington Germander or Teucrin A substance ethanol extract acetone extract teucrinA
dose 2gkg 4gkg 0.5 g k g 0.5 g k g 150mgkg 150mgkg 150mgkg
preplasma ALT,b n treatmenta SFU/mL 2 none 1277,2941 2 none 3747,4176 7 none 2246 f 400 4 PBO 524 f 56d** 5 none 18995~883 5 PBO 215% 32d* 5 PB 3818+45Od*
hepatic necrosisc 2+,4+ 3+,4+ 1.7 % 0.3 0.3 f 0.3d* 2.9f1.0 0.4!~0.3~* 3.0f0.5
a Pretreatments were administered as described in the Experimental Procedures. Values are either for individual mice or are mean & SE. e Hepatic necrosis scores were determined as described in ref 14. Necrosis was primarily localized to the midzonal region of the liver except after PB treatment, where it was primarily localized to the periportal regions. In the highest dose of the ethanol extract, both midzonal and centrilobular necrosis was observed. Significantly different from nonpretreated mice (*P< 0.05; **P< 0.01).
P450 inhibitor, PBO. The marc remaining after the acetone extract of germander caused no evidence of hepatocellular injury in mice, even at twice the dose of the acetone extract (data not shown). Teucrin A, one of the neoclerodane diterpenoids present in the acetone extract, caused midzonal necrosis at 150 mgkg comparable to that of the 0.5 g k g dose of the acetone extract (Figure 3, top panel). The extent of this necrosis also was significantly decreased by PBO (Figure 3, middle panel). Interestingly, the hepatocellular injury was somewhat enhanced by pretreatment of mice with PB based on increases in plasma ALT concentrations (Table l ) , but the necrosis had shifted from the midzonal region to the periportal region (Figure 3, bottom panel). A time course of changes in plasma ALT concentrations compared to changes in hepatic GSH concentrations showed that hepatic GSH most rapidly is depleted from 3 to 6 h after a hepatotoxic dose of teucrin A, while ALT concentrations most rapidly increase from 6 to 12 h after the dose (Figure 4). Pretreatment of mice with BSO, an inhibitor of glutathione synthesis, apparently increased hepatocellular damage caused by teucrin A, as indicated by a rise in 24 h ALT concentrations from 4138 & 365 to 6852 & 652 SFU/mL (P < 0.05;data not shown in Figure 4). Finally, the hepatotoxic effects of tetrahydroteucrin A were compared to those of teucrin A (Figure 5). Whereas teucrin A caused large increases in plasma ALT concentrations 24 h after administration, tetrahydroteucrin A did not elevate serum ALT concentrations above vehicle controls (Figure 5). There also was no observable hepatic necrosis in these mice compared to teucrin A treated animals that sustained 1+ to 3+ midzonal necrosis scores. It should be noted that this group of mice was more resistant to the hepatotoxic effects of teucrin A than the groups used for collection of the data in Table 1, as determined by a limited dosing range study that we normally carry out on the various batches of animals that are purchased.
Discussion The goals of this study were the following: (1)t o establish an animal model for hepatotoxicity observed in humans that was caused by the herbal medicine germander, (2) to determine if furanoneoclerodanes present
in germander caused the same kind of hepatic injury, (3) to investigate the possible role of P450-mediated formation of reactive metabolites in the mechanism of toxicity, and (4) to assess the role of the furan ring of the neoclerodane structure in mediating hepatocellular injury. While this work was in progress, Loeper et al. (8) published results of their studies which clearly demonstrated that mice administered a lyophilized water extract of germander exhibited hepatic necrosis similar to that observed in several individuals in France who had ingested capsules of powdered germander for weight control (3-7). They also found that a fraction of the extract enriched in the neoclerodane diterpenoids was more hepatotoxic than the lyophilate and that P450s of the 3A family were likely involved in activating the furanoneoclerodanes to hepatotoxic metabolites (8). Results of our studies support these findings and extend them. Administration of an ethanol extract of germander caused midzonal necrosis in mice, and midzonal necrosis extending to centrilobular necrosis of the livers at the highest dose (Table 1). An acetone extract enriched in the neoclerodane diterpenoids caused midzonal necrosis at a lower dose, and the extent of this necrosis was significantly decreased by pretreatment of mice with the P450 inhibitor PBO. The marc remaining after extraction of the powedered germander with acetone caused no evidence of liver damage. The primary difference between the results of our studies and those of Loeper et al. (8) was that their water extract lyophilate caused plasma ALT rises similar to ours but at about half the dose that we used, indicating that their extract was a more potent hepatotoxin. One possible reason for this difference is that the diterpenoid content of their germander is higher than that from the germander we harvested at the University of Washington. Teucrin A (Figure l ) , one of the neoclerodane diterpenoids in germander, was found at approximately 4 times the concentration by dry weight in a European source of the germander compared to the Washington germander. A second possible reason is that Loeper et al. (8) boiled their germander at 100 "C for several hours in water prior to preparing their lyophilate, whereas we prepared extracts at close to room temperature (30 "C). Because several of the neoclerodanes present in germander contain structural groups subject to dehydration (alcohols and esters) and hydrolysis (oxetanes and lactones) (9, 10,15j, the heating process may have altered the composition of the terpenoid fraction. We isolated and crystallized teucrin A (Figure 1)from acetone extracts of both Washington and European germander. This furanoneoclerodane diterpenoid was found to cause the same midzonal hepatic necrosis as observed with extracts of the powdered plant material (Table 1, Figure 3, top panel). Evidence that activation of teucrin A by P450 is required for initiation of the hepatocellular damage is provided by results of P450 inhibition by PBO which markedly attenuated the toxic response (Table 1, Figure 3, middle panel). In contrast, pretreatment of mice with the P450 inducer PB modestly enhanced the hepatotoxic response with a shift in the zone of necrosis to the periportal regions of the liver (Table 1; Figure 3, bottom panel). We have observed similar zonal shifts in necrosis caused by other furans after pretreatment with PB (19). Loeper et al. (8) found that the more selective P450 3A inducer, dexamethasone, caused a greater rise in plasma ALT concentrations in mice. We have preliminary evidence that this pretreat-
854 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
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Figure 3. Hepatic necrosis in mice killed 24 h after the oral administration of teucrin A (150 mgkg). Liver sections were prepared by staining with hematoxylin and eosin. Original magnification was 25 x for each sample; following reduction for printing, the final magnification shown is 16.25x . (Top) Approximately 2+ midzonal necrosis after teucrin A, (middle) almost no necrosis after teucrin A and pretreatment with PBO; (bottom) approximately 3+ periportal necrosis after pretreatment with PB.
ment also shifts the zone of necrosis to the periportal region. The reasons for this zonal shift are presently not known.
Evidence that an electrophilic reactive metabolite of teucrin A is formed as a hepatotoxin comes from studies of the effects of GSH depletion. Just prior to a marked
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Chem. Res. Toxicol., Vol. 7, No. 6, 1994 855
Hepatotoxicity of Germander and Teucrin A Plasma ALT HepaticGSH
5000
2
c.
-
4Ooo3000 Q
E
a a
2000
-
1000 -
c
115
8
95
I
cn 75
55
a 0 .c n I
Time Postdosing (hr)
Figure 4. Time courses of plasma ALT concentration changes and hepatic GSH depletion after hepatotoxic doses of teucrin A (200 mgkg) in mice. Error bars are f S E from mean values for 3 mice at each time point. Plus signs indicate significant P < 0.001; differences from control mice (+,P < 0.01;
P
< 0.05). 2000 h
+++,
++,
1
I
There are a couple of caveats that should be remembered. The first is that teucrin A represents only a fraction of the total furanoneoclerodane diterpenoids found in germander (9, 10). Therefore, there may exist other compounds in this class that are more potent hepatotoxins and that may contain other structural moieties that contribute to cell injury. Second, as has been pointed out by Loeper et al. (81, the doses of germander that have been ingested by most individuals who presented with evidence of liver damage were lower than those that were necessary to cause liver injury in mice. Thus, there is the possibility that reactive metabolites of the furan ring, or other structures of the neoclerodane diterpenoids (or even other germander components), may have been involved in generating immunogens that resulted in the cases of human hepatitis caused by germander. Additional investigations along these lines are underway.
Acknowledgment. We wish to thank Dr. M. F. Denton, Mr. D. M. Ewing, and Karen Dorweiler, Department of Botany, University of Washington, for identifying the plant material. We would also like to thank Professor Benjamin Rodriguez, CSIC, Madrid, Spain, for kindly providing an authentic analytical sample of teucrin A, and Dr. S. Ananda Weerawarna of the University of Washington, Department of Medicinal Chemistry, for helpful discussions. This work was supported by NIH Grant GM25418.
References (1) Delaveau, P. (1986)La Germandree petit-chhe (Wild germander). Actual. Pharm. 238, 34-39. (2) Grieve, M. (1959) Wall Germander. In A Modern Herbal (Leyel,
C. F., Ed.) Vol. I, p 352, Hafner Publishing Co., New York. Vehicle
Tetrahydroteucrin A
Teucrin A
Figure 5. Plasma ALT concentrations in mice 24 h after treatment with either the vehicle used for oral intubation (0.5 mL of Tween 80/corn oil/saline, 2:2:6), vehicle plus tetrahydroteucrin A (200 mgkg), or vehicle plus teucrin A (200 mgkg). Error bars are f S E from mean values for 5 mice in each group. Histopathological necrosis was only observed in the teucrin A-treated group and was midzonal (mean necrosis score f SE was 2.1 f 0.4). Significantly different from vehicle and tetrahydroteucrin A-treated mice (++, P < 0.001).
increase in plasma ALT concentrations in mice from 6 t o 12 h after the administration of a hepatotoxic dose of teucrin A (Figure 4), GSH is depleted to about 25% of control values (Figure 4). Loeper et al. (8) used phorone, an agent that is conjugated with GSH, to show that prior GSH depletion enhanced the hepatotoxic effects caused by extracts of germander. Using the GSH synthesis inhibitor BSO, we found that the extent of hepatotoxicity caused by teucrin A also was increased. Finally, and most importantly from a mechanistic standpoint, we found that selective reduction of the furan ring of teucrin A yielded a mixture of epimers of tetrahydroteucrin A (Figure 1) that was not hepatotoxic (Figure 5 ) . This provides strong support for the hypothesis that oxidation of the furan ring structural unit of the neoclerodane diterpenoids is involved in the initiation of hepatocellular injury caused by germander. Several furan-containing compounds are known to cause acute lethal cell injury (20-24), and the mechanism appears to involve cytochrome P450-mediated formation of reactive electrophilic,oxidative products of the furan ring (19, 22, 25-27).
(3) Castot, A., and Larrey, D. (1992) Hepatites observees aux cours
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