Toxicology Letters, 70 (1994) 28 l-290 0 1994 Elsevier Science Ireland Ltd. All rights reserved 0378-4274/94/$07.00

281

TOXLET 02983

Metabolism of benzene and trans, trans- muconaldehyde in the isolated perfused rat liver

V. Lee Grotz, Sungchul Ji, Stanley A. Kline, Bernard D. Goldstein and Gisela Witz

Joint Graduate Program in Toxicology, Rutgers UniversitylUMDNJ-Robert Wood Johnson Medical School

and Environmental Occupational Health Sciences Institute, Piscataway. NJ (USA)

(Received 9 February 1993) (Accepted 12 April 1993)

Key words: Benzene; trans. trans-Muconaldehyde; Liver perfusion

SUMMARY

Perfusate from rat livers perfused with benzene (-0.7-7 x 10m4 M) or trans, trans-muconaldehyde (MUC) (lO-4 M) was extracted and analyzed by reverse-phase HPLC. Based on retention time and co-elution experiments, benzene was found to be metabolized to trans, trans-muconic acid, a urinary ring-opened metabolite of benzene and a major in vivo and in vitro metabolite of MUC. These data demonstrate that benzene ring-opening occurs in the liver. Following perfusion with MUC (a microsomal hematotoxic metabolite of benzene), trans, trans-muconic acid and three other MUC metabolites were detected in the perfusate extract, suggesting that these metabolites would be present in the circulation following metabo- lism of MUC.

INTRODUCTION

The toxicity of benzene, a potent hematotoxin, is generally considered to be de- pendent on its metabolism. The majority of benzene metabolism is known to occur in the liver and result in the formation of potentially toxic ring-closed metabolites (for recent reviews see Refs. 1 and 2). A ring-opened metabolite of benzene, tram, trans-

muconaldehyde (MUC, muconaldehyde), a six-carbon diene dialdehyde, has also been postulated to play a role in the toxicity of benzene [3]. Previous studies from our

Correspondence to: Dr. Gisela Witz, Toxicology Division, Environmental Occupational Health Sciences Institute, 681 Frelinghuysen Road, P.O. Box 1179, Piscataway, NJ 08855-I 179, USA.

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laboratory have shown that microsomal metabolism of benzene results in the forma-

tion of MUC [4] and that low doses of MUC administered intraperitoneally to mice

induce hematotoxic effects similar to those caused by benzene IS]. Muconaldehyde is

metabolized by a mouse liver soluble fraction to the mixed aldehyde carboxylic acid

derivative (CHO-M-COOH) which is subsequently oxidized to trans, trans-muconic

acid (COOH-M-COOH), the corresponding diene diacid derivative [6]. In vivo stud-

ies have shown that tram, trans-muconic acid is a urinary metabolite of both benzene

and MUC [7-lo] lending further support to the hypothesis that MUC is an intermedi-

ate of benzene in vivo.

The site of benzene metabolism to ring-opened metabolites, however, is unknown.

Partial hepatectomy has been shown to have preventive effects against benzene toxic-

ity Ill]. If trans,trhns-muconaldehyde is a benzene metabolite of significance with

respect to the toxicity of benzene, it seems reasonable that the liver would be a major

site of benzene ring-opening. This hypothesis is supported by in vitro studies of ben-

zene metabolism. Latriano et al. demonstrated that benzene is metabolized by mouse

liver microsomes to MUC [4]. Hepatocyte studies by Schrenk and Bock [ 121 have

shown that the liver is a possible site for the metabolism of benzene to ring-opened

metabolites.

The objective of the present studies was to determine whether benzene is metabol-

ized by the liver to ring-opened compounds. A further objective was to determine the

metabolic fate of muconaldehyde in the isolated perfused rat liver. In comparison to

in vitro metabolism, metabolism of benzene and MUC in the isolated perfused liver

may more closely mimic in vivo metabolism of these compounds. The latter studies

were thus performed to help clarify and/or confirm results of previous in vitro studies

of benzene and MUC metabolism [6,12-161.

MATERIALS AND METHODS

Chemicals

HPLC-grade solvents were purchased from Fisher Scientific Company (Fair Lawn,

NJ). Trans, trans-muconic acid (COOH-M-COOH) was purchased from Aldrich

Chemical Company, Inc. (Milwaukee, WI). Trans, trans-muconaldehyde (CHO-M-

CHO) was custom-synthesized by Calbiochem (San Diego, CA) according to the

method of Kossmehl and Bohn [ 171 used previously in our laboratory [3]. 6-Hydroxy- trans. trans-2,4-hexadienoic acid (COOH-M-OH), 6-oxo- tram, trans-hexadienoic acid

(COOH-M-CHO), and 6-hydroxy-trans, trans-2,4-hexadienal (CHO-M-OH) were

synthesized and characterized in our laboratory [l&19]. All other chemicals were of analytical or reagent grade purity and were purchased from Sigma Chemical Co. (St.

Louis, MO).

Animals

Adult male Sprague Dawley and Fisher 344 rats (280-380 g) were purchased from

Charles River (Wilmington, MA). The animals were acclimated for at least 1 week in

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a climate-controlled animal care facility with a 12-h light cycle. Food (Purina Rodent Chow No. 5001) and water were available ad libitum during the acclimation period.

Isolated rat liver perfusion Rats were anaesthetized with sodium pentobarbital (50 mg/kg) prior to surgical

procedures. The livers were isolated and perfused (one-pass; non-recirculating) via the portal vein with Krebs Henseleit bicarbonate buffer (KHB) equilibrated with a 95/5% oxygen/CO, mixture according to the methods of Ji et al. [20]. The flow rate was 35 to 50 ml/min and the temperature was maintained at about 37 + 1°C. Effluent perfusate was collected via the hepatic vein. Venous oxygen tension (measured with a Clark-type platinum electrode immersed in the effluent), portal pressure (measured with a water-filled manometer), and the visual appearance of the livers were moni- tored throughout the perfusion in an effort to assess the relative health of the livers.

Following the control perfusion with KHB alone, the liver was perfused with KHB containing either benzene or MUC. In the benzene experiments, a saturated solution of benzene in KHB (-9 mM) was steadily mixed with control perfusate (KHB) enter- ing the liver via an infusion pump. In initial experiments, the resulting concentration of benzene in the perfusate was approx. 0.7 x 10m4 M. In subsequent experiments, the concentration was increased to -7 x 10e4 M. In the MUC experiments, perfusion was switched from one reservoir containing plain perfusate to another containing MUC at a concentration of 1O-4 M. Effluent perfusate was collected in 45 ml fractions for up to 3 h following the initiation of perfusion.

Perfusate extraction Aliquots (45 ml) of effluent perfusate from both control livers and livers perfused

with solutions containing either benzene or MUC were collected approx. 30-60 min following perfusion. The aliquots were acidified to pH < 3 with 1 M HCL and ex- tracted twice with 2 ~01s. of ether. The ether extracts were pooled and evaporated under N, almost to dryness while being maintained at 37°C in a water bath. The residue was dissolved in methanol (final volume 500@). The methanolic samples were passed through a 45 ,um filter, and aliquots (20-100 ~1) were analyzed by HPLC.

HPLC analysis Separations of benzene or MUC metabolites from perfusate extracts were per-

formed using an HPLC system equipped with a Waters 600E pump, a Waters 484 UV-visible detector and a Maxima 820 integrator. The samples were applied to a Waters reverse-phase Nova-Pak” C,* 4 pm radially compressed column (8 x 100 mm), preceded by a C8 4 pm guard cartridge (Waters, Milford, MA), and eluted isocratically with 1% aqueous acetic acid/methanol (90: 10, v/v) or by gradient elution at a flow rate of 1 ml/min. For gradient elution, 1% aqueous acetic acid (solvent A) was applied to the column for 10 min, followed by a 15 min linear gradient of &IO% methanol (solvent B), and a subsequent 15 min elution with a 90: 10 (v/v) mixture of solvents A and B, respectively. For both gradient and isocratic elutions, the columns

284

were washed with methanol for a minimum of 5 min, returned to the starting condi- tions, and equilibrated for a minimum of 15 min. The wavelength of detection in samples analyzed isocratically was 254 nm and 265 nm for those analyzed by gradient elution. Concentrations of the benzene and muconaldehyde metabolites were calcu- lated from the corresponding integrated chromatogram peak areas of standards of known concentration.

Analysis of extracts from rat livers perfused with 10e4 M MUC was also performed by reverse-phase HPLC using a LiChrosorb RP-18 (5 PM) 25 x 0.46 cm analytical column (E. Merck, Darmstadt, Germany) preceded by a RP-18 Supelco guard col- umn. For the latter, a Spectra Physics HPLC system equipped with a 8800 pump, a 8489 dual’wavelength UV-visible wavelength detector and a 8840 integrator was used. The wavelength of detection was 265 nm. On-line spectra were also recorded using a Perkin-Elmer LC-410 auto Scan Diode Array UV detector. Gradient elution of sam- ples applied to the column was performed as described above.

RESULTS

Isolated rat liver perfused with benzene Using isocratic elution and reverse-phase chromatography, HPLC chromatograms

of extracted perfusate from three isolated rat livers perfused with -7 x 10F4 M ben- zene show a peak present at about 13 min. A representative HPLC chromatogram is shown in Figure 1A. The retention time of this peak is consistent with that of authen- tic tram, trans-muconic acid (COOH-M-COOH). Extracts spiked with authentic COOH-M-COOH show the co-elution of the unknown peak with authentic COOH- M-COOH (Fig. 1B). Using gradient elution, the HPLC chromatograms of the three perfusate extracts from benzene-treated isolated rat livers also showed a peak at the retention time of COOH-M-COOH (about 25 min). A representative HPLC chroma- togram is shown in Figure 1C. Reverse-phase HPLC analysis of extracted control perfusate did not reveal the presence of any material with a retention time similar to that of COOH-M-COOH (representative chromatogram is shown in Fig. 1D). Using gradient conditions, the benzene metabolites, benzene dihydrodiol, hydroquinone, catechol, phenol and benzoquinone, elute at approx. 7, 8, 15,29 and 32 min (data not shown). These retention times and those of the alcohol-acid, aldehyde-alcohol and acid-aldehyde metabolites of MUC (see muconaldehyde perfusion results below) are different from that of the peak identified as COOH-M-COOH in the extracts of perfusate from isolated rat livers perfused with benzene. Based on the above results, the peak at about 13 min (isocratic elution) and 25 min (gradient elution) in perfusate extracts from benzene-treated livers has been identified as COOH-M-COOH.

Based on area under the curve (AUC) data from standards of known concentra- tion, the concentration of COOH-M-COOH in the perfusate extracts was approx. 5-7 ng/20@ (about 3 ng/ml or 2 x lo-’ M in unextracted perfusate). The limit of detection for COOH-M-COOH under the conditions employed (which utilized the maximum detector sensitivity settings) was 2 r&20 ,ul. When the rat liver was perfused with

285

A 35-

30-

CooH-M-COOH (13.7)

25- I

AA

:201 , , , I , > 5 15 25 : .-

30- CooH-M-COOH

I II (24.5)

X-l

25-

C

I 2o 5

I I I I l 15 25 35

COOH-M-COOH (12.3)

1

L B

II I I I I 5 15 25

I I I I I I 5 15 25 i!

Time (min)

Fig. 1. High pressure liquid chromatograms of metabolites formed from benzene in the non-recirculating isolated perfused rat liver. The liver was perfused with -7 x 10e4 M benzene, and perfusate extracts were

prepared and analyzed by HPLC as described in Materials and Methods. (A) Extract of perfusate from liver perfused with benzene and analyzed by isocratic elution. (B) Same as A, and spiked with authentic muconic acid. (C) Extract of perfusate from liver perfused with benzene and analyzed by gradient elution.

(D) Extract of perfusate from untreated liver and analyzed by gradient elution.

benzene at a concentration of -0.7 x 10m4 M, COOH-M-COOH was not detected in the HPLC chromatograms of the perfusate extracts.

Gradient elution of the perfusate extracts also showed the presence of two addition- al peaks (X-l, X-2 - Fig. 1C) not seen in control perfusate. The retention time of one of these peaks (X-l) was 21.6 min. Under the conditions utilized, this retention time was similar to that of authentic COOH-M-OH (21.8 min, data not shown), i.e., the acid-alcohol MUC metabolite detected in mouse liver cytosol incubated with MUC [14-16,181.

Isolated rat liver perfused with tram, trans-muconaldehyde Using reverse-phase HPLC with gradient elution, analysis of extracts from three

isolated rat livers perfused with low4 M MUC showed that MUC is metabolized to several different products. The HPLC chromatogram of one perfusate extract is shown in Figure 2. In this chromatogram, three major peaks (20.3,26.4 and 28.0 min) and five minor peaks (9.5,23.0,24.4,31.3 and 32.8 min) were observed. Similar peaks

286

COOH-M-OH - c COOH-M-COOH

E

lE

Et

z COOH-M-CHO

: 0.01

: a

0 03

s

0

0 10 20 30 40

Time (min)

Fig. 2. High pressure liquid chromatogram of metabolites formed from MUC in the non-recirculating isolated perfused rat liver. The liver was perfused with 1 x 10m4 M MUC, and perfusate extracts were

prepared and analyzed by HPLC as described in Materials and Methods.

were not detected in the HPLC chromatograms of extracted control perfusate (data

not shown).

Based on the results of co-elution experiments, the relative peak retention times and

diode array spectra, the material under the peaks at 20.3,24.4,26.4, and 28.0 min was

identified as the acid-alcohol (COOH-M-OH), aldehyde-alcohol (CHO-M-OH), di-

acid (COOH-M-COOH) and acid-aldehyde (COOH-M-CHO) analogs of muconalde-

hyde, respectively. The retention times and wavelengths of maximum absorption for

these peaks were consistent with the retention times and maximum absorption wave-

lengths for the authentic standards. HPLC chromatograms of the perfusate extract

spiked with authentic COOH-M-OH, COOH-M-COOH and COOH-M-CHO (each

standard added separately to three different aliquots of perfusate extract) showed the

co-elution of the standards with the observed HPLC perfusate extract peaks at about

20,26 and 28 min, respectively. The minor peak seen at 31.3 min had a retention time

consistent with that of authentic MUC. The identity of the minor peaks at 9.5, 23.0

and 32.8 min (X-l, X-2 and X-3, respectively) is not known. The retention time of the

peak at 32.8 min, however, is similar to that of an unknown peak also seen in the

perfusate extract from isolated rat livers perfused with -7 x 10e4 M benzene.

281

The HPLC chromatogram of extracted perfusate from a second liver was similar to the one just described (data not shown). The HPLC chromatograms of extracted perfusate from a third liver showed one major peak at about 26 min corresponding to COOH-M-COOH and four minor peaks at about 24, 28, 31 and 33 min (data not shown). The retention times for the peaks at about 24 and 28 min are consistent with the retention times of the aldehyde-alcohol and acid-aldehyde analogs of MUC, while the retention time for the 31 min peak is consistent with that of MUC.

The concentrations of the MUC metabolites seen in perfusate from different livers varied. Based on AUCs for authentic standards, the concentration of COOH-M- COOH (the most abundant material detected) in 30-60 min perfusate samples was 0.4-5.6 x lo-’ M. Hence, a significant proportion of MUC was quickly metabolized to COOH-M-COOH. Based on the AUCs for authentic standards, the range of con- centrations observed in the perfusate was O-4.1 x 10e5 M for COOH-M-OH, 1 .l- 2.7 x 10e6 M for CHO-M-OH and 0.02-3.8 x 10m6 M for COOH-M-CHO. Based on the AUCs for authentic MUC standards, the concentration of unmetabolized MUC was approx. l-7 x lo-’ M in all samples assayed.

DISCUSSION

The results of the present in vitro studies indicate that the liver is a site of in vivo benzene metabolism to ring-opened products. This conclusion is based on experi- ments which showed the formation of COOH-M-COOH following perfusion of the isolated rat liver with -7 x 10e4 M benzene. Although COOH-M-COOH was not detected in perfusate extracts following perfusion with -0.7 x 10m4 M benzene (a lo-fold decrease in benzene concentration), the amount of COOH-M-COOH, if pres- ent, was likely below the limit of detection. The latter assumption is based on the fact that the concentration of COOH-M-COOH achieved in the perfusate following per- fusion with -7 x 10m4 M benzene was approx. 2.5-times that of the detection limit. A lo-fold decrease in dose may have led to a similar decrease in the concentration of COOH-M-COOH in the perfusate, which would be below our limit of detection.

Similarly, any MUC formed and present in the perfusate would also likely have been below our detection limit. Following perfusion with 10m4 M MUC in the isolated rat liver, the concentration of COOH-M-COOH in the exiting perfusate was approx. 300-times that of unmetabolized MUC in all samples analyzed. If MUC is formed in the isolated rat liver perfused with -7 x 10m4 M benzene and metabolized in a manner analogous to that seen in the MUC perfusion studies, its concentration would be sufficiently low as to be below our detection limit. As a consequence, the use of the one-pass (non-recirculating) isolated rat liver perfusion system to fully characterize the metabolism of benzene has limitations. Recirculation of the perfusate would pre- sumably result in increased concentrations of some metabolites in the final perfusate, thus increasing the possibility of their detection.

Phenol, the major metabolite of benzene was not detected in the perfusate extracts analyzed. It is possible that phenol was lost during evaporation of the ether extract or

288

that it was further metabolized to water-soluble conjugates not extracted into the

ether phase.

In addition to establishing that the liver is a site of benzene metabolism to ring-

opened products, our results add further support to the hypothesis that the highly

reactive a&unsaturated aldehyde, MUC, is a benzene metabolite and the putative

precursor of COOH-M-COOH found in the urine of humans and several laboratory

species exposed to benzene [l]. Muconic acid was found in perfusate extracts taken

from isolated rat livers perfused with either 10m4 M MUC or -7 x 10m4 M benzene.

Additionally, in the benzene experiments, a metabolite was detected with a retention

time consistent with that of the acid-alcohol MUC metabolite seen in the MUC

isolated liver perfusion studies. The amelioration of benzene toxicity observed with

partial hepatectomy [l l] may thus be a consequence of a diminished metabolism of

benzene to not just potentially toxic ring-closed metabolites but also ring-opened

metabolites.

The metabolism of MUC by purified yeast alcohol and aldehyde dehydrogenases,

rat hepatocytes, and mouse liver cytosol has been reported to result in various oxi-

dized and reduced metabolites [14-16,181. In rat hepatocytes incubated for 30 min

with MUC [15], the major metabolites were COOH-M-COOH and COOH-M-OH

and the minor metabolites were COOH-M-CHO and CHO-M-OH. The present iso-

lated liver perfusion studies confirm that both COOH-M-COOH and COOH-M-OH

are predominant MUC metabolites produced in the liver and are found in the exiting

perfusate. The more reactive MUC metabolites, COOH-M-CHO and CHO-M-OH,

(i.e., cr&unsaturated aldehydes) were also confirmed as hepatic metabolites of MUC.

The presence of the latter metabolites in the perfusate almost certainly means that

these metabolites would be released to the circulation following in vivo metabolism of

MUC. The likely release of these MUC metabolites would not necessarily be predict-

ed from previous in vitro or in vivo studies. The present findings are of importance

since COOH-M-CHO and CHO-M-OH, and not just MUC, may play a role in the

toxicity of benzene. As a chemical class, @-unsaturated aldehydes are highly reactive

electrophiles which are direct-acting alkylating agents. Hence, the release to the

bloodstream of any ring-opened metabolite of benzene with an @-unsaturated alde-

hyde functional group may have significance with respect to the overall mechanism of

benzene toxicity.

It is interesting to note that MUC perfusion of livers from the same batch of

animals did not result in perfusates with quantitatively similar metabolic profiles. The

perfusate samples studied were aliquots representing similar, although not exactly the

same, time fractions. No striking differences were noted in the relative health of the

livers during the perfusions as measured by oxygen uptake, appearance and portal

pressure. The apparent differences in the metabolic profiles may be the result of

differential metabolic rates by the different livers due to differential concentrations of

the metabolizing enzymes.

289

ACKNOWLEDGEMENT

This work was supported by NIH Grant ES02558 and NIEHS Center Grant ES05022.

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