InternationalArchives of
Int Arch Occup Environ Health 44, 117-125 (1979)
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© Springer-Verlag 1979
Influence of Phenobarbital on Xylene Metabolism in Man and Rats Alois David', Jan Flek, Emil Frantik, Ivan Gut, and Vaclav
edivec
Institute of Hygiene and Epidemiology, Center of Industrial Hygiene and Occupational Diseases, CSSR-100 42 Prague 10, Srobarova 48, Czechoslovakia
Summary Retention of inhaled m-xylene vapors and the amount of the excreted metabolite, i e , conjugated m-methylbenzoic acid (MBA), was measured in five volunteers before and after hepatic microsomal enzyme induction by phenobarbital During exposure to m-xylene vapors at a concentration of 400 mg/m3 air, phenobarbital pretreatment did not affect either retention of xylene vapors or the amount of excreted MBA. Higher concentrations of m-xylene were studied in rats At 400 and 800 mg/m 3 , respectively, the result was the same as in humans, i e , the amount of excreted MBA did not differ in pretreated and control animals A rise in the concentration of m-xylene vapors to 2000 and 4000 mg/m3 , respectively, increased fourfold the amount of excreted MBA in phenobarbital pretreated rats compared with control rats. It is assumed that during inhalation of low concentrations of xylene vapors, the normal biotransformation capacity of the liver is sufficient to metabolize the total portion of the absorbed substance entering the liver and, therefore, enzyme induction does not increase the amount of the metabolite formed Only at high concentrations does the amount of the supplied xenobiotic surpass the normal biotransformation capacity of the liver and, in this case, can the higher capacity, due to enzyme induction, assert itself. Key words: Phenobarbital administration Xylene retention and metabolism
Hepatic enzyme induction -
It is well known that some drugs or environmental xenobiotics may induce microsomal enzyme activity The discovery of this property has largely improved the understanding of the interactions between xenobiotics and the organism and deserves further attention Despite increasing interest in the clinical implications of microsomal enzyme induction in the metabolism of various therapeutics 1 Present address: World Health Organization, CH-1211 Geneva 27, Switzerland Offprint requests to: A David, M D (address see above)
0304-0131/79/0044/0117/$ 1 80
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(Gillette and Mitchell, 1975), there appears little knowledge on its effect under conditions of industrial exposure to chemicals (Fouts and Gut, 1978) Two questions are of prime importance in occupational health: in which way does enhanced enzymatic biotransformation activity affect the total balance of absorption, metabolism, and excretion of xenobiotics, and/or how does a changed enzyme activity influence the toxic effect of a substance. This paper is concerned with the first problem in studying the effect of phenobarbital pretreatment on m-xylene retention in the lungs and the excretion of its metabolite by urine in man and rats.
Material and Methods A Human Exposure 1 Induction of Microsomal Enzyme Activity Five healthy male volunteers, aged 46 to 55 years, were given phenobarbital orally (Phenobarbital Spofa) in an amount slightly exceeding 2 mg/kg/day, in a single daily dose at night, for 11 days According to their body weight, two subjects received 150 mg and three subjects 200 mg phenobarbital daily In the last month before the experiments and during the study they did not receive any drug medication. On the 12th day the subjects were exposed to m-xylene vapors at a concentration of 400 mg/m 3 for 8 h. During the treatment by phenobarbital all the volunteers were inhibited, somnolent, having the feeling of dizziness, and one of them developed an itching small papular exanthema on the volar side of the arms After withdrawal of medication, the symptoms and signs disappeared within a few days. 2 Test on Microsomal Enzyme Induction In three subjects, the plasma antipyrine half-life was determined before the administration of phenobarbital and 36 h after the cessation of this treatment Antipyrine, 1 g, was given by mouth in the morning, after overnight fasting; 3, 5, 7, and 9 h thereafter, venous blood samples were taken into heparinized vials Antipyrine concentrations in plasma were determined according to Brodie et al (1949) The values measured were plotted in a semilogarithmic graph and from the fitted straight line the biological half-life of antipyrine was determined. 3 Exposure to M-xylene Vapors All experimental subjects were exposed to a stable concentration of m-xylene vapors for 8 h in our laboratory The concentration was automatically analyzed at 5-min intervals by gas chromatography In accordance with the height of the just recorded peak, the vapor concentration was automatically adjusted to the required level (Sedivec et al , 1974) The mean concentration throughout the experiment was 400 mg xylene/m3 air, with maximum deviations ± 5 % 400 mg/m3 is twice the Czechoslovak wholeshift maximum allowable concentration (MAC = 200mg/m 3). On the first exposure, two individuals (D, ) had just finished 11 days of phenobarbital treatment, while three other volunteers (Fl, Fu, M) were without premedication Six weeks after the first exposure (when the effect of induction disappeared) the roles were reversed and the individuals Fl, Fu, and M were pretreated with phenobarbital Thus, each experimental subject served as its own control Exposure to m-xylene started always at 8 a m and terminated at 4p.m. In the period between the 1st and 3rd h of exposure and again between the 5th and 7th h of exposure, retention of m-xylene vapors in the lungs were determined by measuring the difference between the mean vapor concentrations in the inspired and expired air (gedivec and Flek, 1976 a) The main metabolite of m-xylene, methylhippuric acid, i e , m-toluric acid, was determined in the urine The urine was subjected to alkaline hydrolysis in which mmethylhippuric acid was decomposed to m-methylbenzoic acid, i e , m-toluic acid (further
Phenobarbital on Xylene Metabolism
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MBA) After acidification of the reaction mixture, MBA was extracted into ethyl acetate, converted by diazomethane to its methylester and determined by gas chromatography (Flek and Sedivec, 1973). From the beginning of the exposure all the urine excreted was collected for 24 h. Throughout the exposure and during 6 h after its termination urine was sampled at 2-h intervals; the last sample overnight corresponded to a 10-h interval. B Experiments with Rats 1 Induction of Microsomal Enzyme Activity Experiments were performed on Wistar strain
derived white male rats, weight about 250 g, fed on DOS 2b diet (natural nutrients enriched with vitamins and minerals in optimal proportions for rats, 3 41 kcal/g, 32 % net weight proteins, 5.6 % fat, and 48 % carbohydrates) The rats were given sodium phenobarbital orally in a dose of 50 mg/kg/day for three successive days. 2 Exposure to M-xylene Vapors Forty-eight hours after the last dose of phenobarbital, the animals were exposured for 6 h to m-xylene vapors at concentrations of 400, 800, 2000, and 4000 mg/m 3 air, respectively Six rats were used for each concentration. During the exposure and 18 h afterwards the animals (two per cage) were placed in a ground joint glass metabolic cage Kavalier of about 61 volume The air, with a known concentration of m-xylene vapors, was supplied at a flow rate of 41 per min This atmosphere was prepared by passing fresh air through an evaporator into which a measured volume of liquid m-xylene was injected 10 times per min by a piston micropump (Frantik and Kratochvile, 1976) The vapor concentration in the resultant gaseous mixture was checked every 2 h by interferometer In none of the measurements did it deviate more than 10 % from the required value. Urine, free of feces, was collected during the exposure (6 h) and for further 18 h after the exposure At the end of each sampling period, the animals were forced to urinate by palpation on the belly and the walls of the exposure cage were rinsed with distilled water Loss of urine was so reduced to a minimum. MBA was determined in the urine of the animals by the same method as for human urine.
Results Antipyrine Half-life Prior to administration of phenobarbital the antipyrine half-life was 12 5 h in one individual (Fu) and 17 h in the remaining two individuals (Fl, M) After the treatment, antipyrine half-life was reduced to 10 25 h in all three subjects. Retention of M-xylene Vapors in the Lungs The retention of xylene in the lungs of all subjects was in the range of 51 0 to 67.5 %, mean 57 6 %, in the subjects with phenobarbital pretreatment, and in the range 49 8 to 72 8 %, mean 58 9 %, without the phenobarbital pretreatment The difference is not statistically significant There was also no difference between morning and afternoon measurements Thus, pretreatment with phenobarbital did not increase the retention of m-xylene vapors. Excretion of M-methylbenzoic Acid (MBA) in Humans The amount of excreted MBA is plotted in Figs 1 and 2 Figure 1 shows the mean excretion curve in all 5 volunteers without and with the phenobarbital pretreat-
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mg/h
:u 150 0o o
Fig 1 Mean values of the excreted amount of conjugated m-methylbenzoic acid in five male volunteers without (empty circles) and with (black dots) pretreatment by phenobarbital 2mg/kg/day for 11 days Urine was collected in seven 2-h intervals and in the last 10-h interval, total 24 h
o 100 .0
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4
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8
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Fig 2 Amount of conjugated m-methybenzoic acid excreted 1.0
c , g
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, a; x L
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Fig 2 Amount of conjugated m-methylbenzoic acid excreted
by the individuals during 8-h exposure (black symbols) and oduring 24-h of collecting urine (empty symbols) and the averages of the groups during 8 h (hatched bottom part of the columns) and during 24 h (whole columns) without (C) and with (P) phenobarbital pretreatment
P
ment Figure 2 shows the amount of MBA excreted during 8 h of exposure and during 24 h from the beginning of the exposure; symbols corresponding to each individual and mean values for the whole group are given It is evident that phenobarbital pretreatment had no effect on the amount of excreted m-xylene metabolite. Excretion of M-methylbenzoic Acid (MBA) in Rats The amount of MBA excreted by rats is given in Figure 3 as the arithmetic mean of the values calculated per rat The exposure to concentrations of 400 and 800 mg m-xylene/m 3 air, respectively, gave similar results as in humans, i e , the difference between the amounts excreted by control and by phenobarbital pretreated rats was not statistically significant The twofold rise in the concentra-
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mg 90 80 Z 70 o
u 60 O 50
Fig 3 Arithmetic means of the amount of m-methylbenzoic acid calculated per rat, excreted during 6 h of exposure (hatched bottom part of columns) and during 24 h of collecting urine (whole columns) Vertical bars indicate 95% confidence intervals for means Asterisks indicate statistically significant differences between values before (C) and after (P) phenobarbital pretreatment (P< 005)
40
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Fig 4 Amount of m-methylbenzoic acid (Y) excreted per rat during 6 h of inhalation as a function of m-xylene concentration in inhaled air (X) Experimental points are the mean values for untreated (C-empty circles) and pretreated (P-black dots) rats
tion of xylene vapors (from 400 to 800 mg/m 3 ) doubled the amount of the excreted metabolite. However, a further increase in the concentration of xylene vapors (2000 and 4000 mg/m3 ) revealed a striking difference The control rats excreted during the exposure a similar amount of the metabolite regardless of the rising concentration of xylene in the air; merely the amount of MBA excreted during 24 h increased The increase, however, was not proportional to the rise of xylene concentration in the air (if it is assumed that the retained amount should be proportional to the product of the concentration and the duration of exposure).
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20 40 g/m 3 m-xylene O4 08 Fig 5 Mean amount of m-methylbenzoic acid excreted by rats during 6 h of exposure, expressed as the percent of the total amount excreted during 24 h (= 100 %) Denoted as in the foregoing figures
On the other hand, the amount of MBA excreted by phenobarbital pretreated rats increased during the exposure as well as during 24 h The excreted amount in this group corresponded well to the exponential course according to the equation y= 53 ( 1-e-0 35 X), where x is the concentration of xylene in g/m 3 and y is the
amount of MBA excreted per rat during 6 h of exposure in mg The corresponding equation for untreated rats is y = 13 (1-e-l' 4 x), but the agreement with the measured values is not as good (Fig 4). Figure 5 expresses the amount of MBA excreted during 6 h of exposure as the per cent of the total amount excreted during 24 h A statistical analysis confirmed the significance of the difference between the control and pretreated animals at the concentrations of 2000 and 4000 mg/m 3; moreover there was also a significant difference from the values obtained at 800 mg/m 3.
Discussion The study was primarily designed to investigate a possible impact of changes in the organism's metabolic activity on results of biological exposure tests, used for evaluating the level of industrial exposure to toxic chemical substances and based on the determination of corresponding metabolites of the substances in urine. When examining subjects exposed under entirely identical conditions, a certain variability of results is always found This can be seen also in our small human group (see Fig 2) As the cause of the variability, differences in the extent of lung ventilation and in the degree of the retention and metabolism of the substance are considered It is, therefore, of importance to know the factors that are responsible for the degree of retention-whether they are predominantly physico-chemical
Phenobarbital on Xylene Metabolism
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parameters of the retention compartment of the body or the biological component, i e , a different ability to metabolize xenobiotics. Xylene is suitable for such a study because it is metabolized by microsomal enzymes in the liver almost exclusively to a single metabolite-conjugated mmethylbenzoic acid, which is not physiologically present in urine. Phenobarbital, the most widely used experimental inducer with a broad spectrum, served for inducing enzyme activity Phenobarbital is also a frequent constituent of various drugs (hypnotics and analgesics) This was just the required situation: to see whether occasional therapy with drugs capable of enzyme induction affects the amount of retained xylene and/or the excreted metabolite. At the same time phenobarbital induction represented a model of physiologically high enzyme activity or of increased activity induced by industrial chemicals. In our human experiments, doses of phenobarbital were used having a tolerable hypnotic effect (though unpleasant and making, e g , car driving impossible) and known to induce enzyme activity even after some days of administration (Conney, 1967 ; Cuccinell et al , 1965 ; Vesell and Page, 1969) We have also proved this effect in three subjects by measuring antipyrine half-life The effect corresponded to data in the literature (Vesell and Page, 1969) The second exposure to xylene was repeated 6 weeks after cessation of phenobarbital administration, i e , after a sufficiently long period for the increased activity to return to normal level (MacDonald et al , 1969). Phenobarbital pretreatment of humans affected neither m-xylene retention in lungs nor the amount of the excreted metabolite in urine Such was the case in animals exposed to similar concentrations However, in rats exposed to severalfold higher xylene concentrations the amount of excreted metabolite (MBA) was markedly increased by phenobarbital pretreatment Lack of inducing effect at exposure levels comparable to the human exposure indicates that under such conditions even the biotransformation capacity of the control rat's liver is sufficient for complete metabolism of inflowing xylene (extraction ratio close to 1) Any increase in xenobiotic metabolizing capacity appears therefore superfluous at this concentration level The limiting factor in the total balance of the xenobiotic metabolism is the concentration of xylene in blood, i e , the amount of xylene entering the liver. The situation differs if the supplied amount of xylene exceeds the physiological metabolic capacity of the liver In such a case, this capacity becomes the limiting factor That is probably why at high xylene concentrations in the air, the amount of the metabolite excreted during the exposure by control animals (Fig. 3, concentrations 2 and 4 g/m3 ) was not higher than at lower concentrations. However, the total amount of MBA excreted during 24 h was somewhat enhanced Apparently at a high concentration level, more xylene is retained in the whole organism The desaturation phase after termination of exposure is thus longer and xylene is available for biotransformation for a longer period of time and in a larger quantity. If the biotransformation capacity of the liver is increased by microsomal enzyme induction it appears capable of metabolizing quantitatively the supplied xenobiotic even at high xylene concentrations The exponential relations between m-xylene concentration in the air and MBA excreted indicate that the maximum
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amount of MBA one animal was able to form within 6 h of exposure increased that at higher xylene from 13 mg to 53 mg due to the induction They also indicate excreted during metabolite the of amount the concentrations the increase in be reached) would value asymptotic an of % 90 , e (i exposure would virtually stop 3 rats Thus, 3 pretreated in g/m 6 6 at and at 1 6 g xylene/m air in untreated the given under capacity metabolic in increase according to these results the quantitative This fourfold about is induction conditions of phenobarbital microsomal relation need not apply to other conditions, as induction of hepatic on a dependent markedly quantitatively and enzyme activities are qualitively (including variety of factors, which include the nature of the inducing agents differences. sex dose levels) and species and lead to enhanced The present study found that enzymatic induction does not the inspired air in concentration its unless metabolism of the absorbed xylene that this important is it practice, health exceeds a certain limit For occupational in the xylene for MAC accepted generally limit lies for man and rat above the 3 as mg/m 800 around apparently is rats working atmosphere The limit for excreted of cent (per evaluation of method one indicated by the observation that the mean values, MBA-Fig 5) showed statistically significant differences of In some significance no revealed values whereas tests of differences in absolute German 3), mg/m 650 (approx ppm 150 countries MAC are near this value: Japan 3 in Czechoslovakia the average concen), ppm (870 mg/m Federal Republic 200 3 (less than 50 ppm), but the ceiling mg/m tration for the whole shift is only 200 may exceed the wholeconcentration in value up to which occasional deviations 3 Limits, 1977). Exposure (Occupational mg/m shift time weighted average is 1000 xylene were for liver human the If the biotransformation capacity of known) then not is course, of (which, liver proportional to the capacity of the rat 3 (nearly mg/m 800 of concentration to at a whole-shift xylene exposure up of excreted MBA ppm) the exposure tests based on monitoring the amount 200 concentrations higher At results correct (gedivec and Flek, 1976 b) would give in the concentration xylene the to less metabolite would arise than corresponds high spontaneous its or induction by air A hepatic metabolic activity enhanced also result as (and metabolized of level could probably increase also the amount the affect could difference this whether of retained) xylene The question remains onset of toxic clinical changes. amount Hence, there is no doubt that, e g , attempts to calculate the absorbed acute in metabolite excreted the of an organic solvent from the amount of blood the cases such in because inhalation poisonings are burdened with errors derived amount is contains very high concentrations of the substances and the turnover of the whole the of balance decidedly lower than in reality (in the primarily biotransformation, without substance the percentage that is eliminated levels). by breath, increases at high dose been tested The human biotransformation capacity for m-xylene has not recent found have we However, either experimentally or in field measurements, air the in vapors styrene of concentration information about the relation of high the From 1976) , al et m 6 (Engstr urine in to the concentrations of mandelic acid a styrene concentration figures reproduced by these authors it appears that up to 3 excretion of mandelic acid increases linearly, but this of 150 ppm, i e , 600 mg/m ,
Phenobarbital on Xylene Metabolism
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does not apply to higher concentrations Corresponding to 150 ppm styrene are approximately 3 g mandelic acid/g creatinine, to 300 ppm styrene only 5 g The authors do not explain this difference; in our opinion the limit of the biotransformation capacity of the liver could have taken part in it. Finally, it should be kept in mind that the quantitative relations in the
excretion of metabolites after gastrointestinal absorption of toxic substances may differ because in this case the whole absorbed amount enters the liver directly and the concentration of the substance in the portal vein surpasses the metabolic capacity of the liver These conditions, however, exceed the usual occupational health considerations.
References Brodie, B B , Axebrod, J , Soberman, R , Levy, B B : The estimation of antipyrine in biological material J Biol Chem 179, 25-29 (1949) Conney, A H : Pharmacological implications of microsomal enzyme induction Pharmacol Rev. 19, 317-366 (1967) Cuccinell, S A , Sansur, M , Burns, J J : Drug interactions in man Lowering effect of phenobarbital on plasma levels of bishydrocoumarin (Dicumarol) and diphenylhydantoin (Dilantin) Clin Pharmacol Ther 6, 420-429 (1965) Engstr6 m, K , Hark 6nen, H , Kalliokoski, P , Rantanen, J : Urinary mandelic acid concentration after occupational exposure to styrene and its use as a biological exposure test Scand J. Work Environ Health 2, 21-26 (1976) Flek, J , Sedivec, V : Determination of toxic substances and their metabolites in biological fluids by gas chromatography VII Toluric or Toluic acids in urine Collect Czech Chem. Commun 38, 1754-1759 (1973) Fouts, J R , Gut, I : Industrial and environmental xenobiotics In vitro versus in vivo biotransformation and toxicity Amsterdam, Oxford: Excerpta medica 1978 Frantik, E , Kratochvile, K : Inhalation exposure apparatus with total evaporation of micropump delivered liquids In: Adverse effects of environmental chemicals and psychotropic drugs, Vol 2, pp 321-324 Amsterdam: Elsevier 1976 Gillette, J R , Mitchell, J R : Concepts in biochemical pharmacology, Part 3 Berlin, Heidelberg, New York: Springer 1975 Mac Donald, M G , Robinson, D S , Sylvester, D , Jaffe, J J : The effects of phenobarbital, chloral betaine and glutethimide administration on warfarin plasma levels and hypoprothrombinemic responses in man Clin Pharmacol Ther 10, 80-84 (1969) Occupational exposure limits for airborne toxic substances Occupational safety and health series, No 37 Geneva: International Labour Office 1977 Sedivec, V , Flek, J : The absorption, metabolism and excretion of xylenes in man Int Arch. Occup Environ Health 37, 205 217 (1976 a) Sedivec, V , Flek, J : Exposure test for xylenes Int Arch Occup Environ Health 37, 219-232 (1976 b) Sedivec, V , Flek, J , MrAz, M : Preparation of an atmosphere with defined content of vapours of studied substances Prac L6k 26, 48-53 (1974) (in Czech) Vesell, E S , Page, J G : Genetic control of phenobarbital induced shortening of plasma antipyrine half-lives in man J Clin Invest 48, 2202-2209 (1969) Received January 29, 1979 / Accepted March 28, 1979
Int Arch Occup Environ Health 44, 117-125 (1979)
}(*U Il)litionl 14 ;rliild
>nlllll
© Springer-Verlag 1979
Influence of Phenobarbital on Xylene Metabolism in Man and Rats Alois David', Jan Flek, Emil Frantik, Ivan Gut, and Vaclav
edivec
Institute of Hygiene and Epidemiology, Center of Industrial Hygiene and Occupational Diseases, CSSR-100 42 Prague 10, Srobarova 48, Czechoslovakia
Summary Retention of inhaled m-xylene vapors and the amount of the excreted metabolite, i e , conjugated m-methylbenzoic acid (MBA), was measured in five volunteers before and after hepatic microsomal enzyme induction by phenobarbital During exposure to m-xylene vapors at a concentration of 400 mg/m3 air, phenobarbital pretreatment did not affect either retention of xylene vapors or the amount of excreted MBA. Higher concentrations of m-xylene were studied in rats At 400 and 800 mg/m 3 , respectively, the result was the same as in humans, i e , the amount of excreted MBA did not differ in pretreated and control animals A rise in the concentration of m-xylene vapors to 2000 and 4000 mg/m3 , respectively, increased fourfold the amount of excreted MBA in phenobarbital pretreated rats compared with control rats. It is assumed that during inhalation of low concentrations of xylene vapors, the normal biotransformation capacity of the liver is sufficient to metabolize the total portion of the absorbed substance entering the liver and, therefore, enzyme induction does not increase the amount of the metabolite formed Only at high concentrations does the amount of the supplied xenobiotic surpass the normal biotransformation capacity of the liver and, in this case, can the higher capacity, due to enzyme induction, assert itself. Key words: Phenobarbital administration Xylene retention and metabolism
Hepatic enzyme induction -
It is well known that some drugs or environmental xenobiotics may induce microsomal enzyme activity The discovery of this property has largely improved the understanding of the interactions between xenobiotics and the organism and deserves further attention Despite increasing interest in the clinical implications of microsomal enzyme induction in the metabolism of various therapeutics 1 Present address: World Health Organization, CH-1211 Geneva 27, Switzerland Offprint requests to: A David, M D (address see above)
0304-0131/79/0044/0117/$ 1 80
118
A David et al.
(Gillette and Mitchell, 1975), there appears little knowledge on its effect under conditions of industrial exposure to chemicals (Fouts and Gut, 1978) Two questions are of prime importance in occupational health: in which way does enhanced enzymatic biotransformation activity affect the total balance of absorption, metabolism, and excretion of xenobiotics, and/or how does a changed enzyme activity influence the toxic effect of a substance. This paper is concerned with the first problem in studying the effect of phenobarbital pretreatment on m-xylene retention in the lungs and the excretion of its metabolite by urine in man and rats.
Material and Methods A Human Exposure 1 Induction of Microsomal Enzyme Activity Five healthy male volunteers, aged 46 to 55 years, were given phenobarbital orally (Phenobarbital Spofa) in an amount slightly exceeding 2 mg/kg/day, in a single daily dose at night, for 11 days According to their body weight, two subjects received 150 mg and three subjects 200 mg phenobarbital daily In the last month before the experiments and during the study they did not receive any drug medication. On the 12th day the subjects were exposed to m-xylene vapors at a concentration of 400 mg/m 3 for 8 h. During the treatment by phenobarbital all the volunteers were inhibited, somnolent, having the feeling of dizziness, and one of them developed an itching small papular exanthema on the volar side of the arms After withdrawal of medication, the symptoms and signs disappeared within a few days. 2 Test on Microsomal Enzyme Induction In three subjects, the plasma antipyrine half-life was determined before the administration of phenobarbital and 36 h after the cessation of this treatment Antipyrine, 1 g, was given by mouth in the morning, after overnight fasting; 3, 5, 7, and 9 h thereafter, venous blood samples were taken into heparinized vials Antipyrine concentrations in plasma were determined according to Brodie et al (1949) The values measured were plotted in a semilogarithmic graph and from the fitted straight line the biological half-life of antipyrine was determined. 3 Exposure to M-xylene Vapors All experimental subjects were exposed to a stable concentration of m-xylene vapors for 8 h in our laboratory The concentration was automatically analyzed at 5-min intervals by gas chromatography In accordance with the height of the just recorded peak, the vapor concentration was automatically adjusted to the required level (Sedivec et al , 1974) The mean concentration throughout the experiment was 400 mg xylene/m3 air, with maximum deviations ± 5 % 400 mg/m3 is twice the Czechoslovak wholeshift maximum allowable concentration (MAC = 200mg/m 3). On the first exposure, two individuals (D, ) had just finished 11 days of phenobarbital treatment, while three other volunteers (Fl, Fu, M) were without premedication Six weeks after the first exposure (when the effect of induction disappeared) the roles were reversed and the individuals Fl, Fu, and M were pretreated with phenobarbital Thus, each experimental subject served as its own control Exposure to m-xylene started always at 8 a m and terminated at 4p.m. In the period between the 1st and 3rd h of exposure and again between the 5th and 7th h of exposure, retention of m-xylene vapors in the lungs were determined by measuring the difference between the mean vapor concentrations in the inspired and expired air (gedivec and Flek, 1976 a) The main metabolite of m-xylene, methylhippuric acid, i e , m-toluric acid, was determined in the urine The urine was subjected to alkaline hydrolysis in which mmethylhippuric acid was decomposed to m-methylbenzoic acid, i e , m-toluic acid (further
Phenobarbital on Xylene Metabolism
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MBA) After acidification of the reaction mixture, MBA was extracted into ethyl acetate, converted by diazomethane to its methylester and determined by gas chromatography (Flek and Sedivec, 1973). From the beginning of the exposure all the urine excreted was collected for 24 h. Throughout the exposure and during 6 h after its termination urine was sampled at 2-h intervals; the last sample overnight corresponded to a 10-h interval. B Experiments with Rats 1 Induction of Microsomal Enzyme Activity Experiments were performed on Wistar strain
derived white male rats, weight about 250 g, fed on DOS 2b diet (natural nutrients enriched with vitamins and minerals in optimal proportions for rats, 3 41 kcal/g, 32 % net weight proteins, 5.6 % fat, and 48 % carbohydrates) The rats were given sodium phenobarbital orally in a dose of 50 mg/kg/day for three successive days. 2 Exposure to M-xylene Vapors Forty-eight hours after the last dose of phenobarbital, the animals were exposured for 6 h to m-xylene vapors at concentrations of 400, 800, 2000, and 4000 mg/m 3 air, respectively Six rats were used for each concentration. During the exposure and 18 h afterwards the animals (two per cage) were placed in a ground joint glass metabolic cage Kavalier of about 61 volume The air, with a known concentration of m-xylene vapors, was supplied at a flow rate of 41 per min This atmosphere was prepared by passing fresh air through an evaporator into which a measured volume of liquid m-xylene was injected 10 times per min by a piston micropump (Frantik and Kratochvile, 1976) The vapor concentration in the resultant gaseous mixture was checked every 2 h by interferometer In none of the measurements did it deviate more than 10 % from the required value. Urine, free of feces, was collected during the exposure (6 h) and for further 18 h after the exposure At the end of each sampling period, the animals were forced to urinate by palpation on the belly and the walls of the exposure cage were rinsed with distilled water Loss of urine was so reduced to a minimum. MBA was determined in the urine of the animals by the same method as for human urine.
Results Antipyrine Half-life Prior to administration of phenobarbital the antipyrine half-life was 12 5 h in one individual (Fu) and 17 h in the remaining two individuals (Fl, M) After the treatment, antipyrine half-life was reduced to 10 25 h in all three subjects. Retention of M-xylene Vapors in the Lungs The retention of xylene in the lungs of all subjects was in the range of 51 0 to 67.5 %, mean 57 6 %, in the subjects with phenobarbital pretreatment, and in the range 49 8 to 72 8 %, mean 58 9 %, without the phenobarbital pretreatment The difference is not statistically significant There was also no difference between morning and afternoon measurements Thus, pretreatment with phenobarbital did not increase the retention of m-xylene vapors. Excretion of M-methylbenzoic Acid (MBA) in Humans The amount of excreted MBA is plotted in Figs 1 and 2 Figure 1 shows the mean excretion curve in all 5 volunteers without and with the phenobarbital pretreat-
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mg/h
:u 150 0o o
Fig 1 Mean values of the excreted amount of conjugated m-methylbenzoic acid in five male volunteers without (empty circles) and with (black dots) pretreatment by phenobarbital 2mg/kg/day for 11 days Urine was collected in seven 2-h intervals and in the last 10-h interval, total 24 h
o 100 .0
E so 50
4
12
8
16
20
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'G 15 .o o N
Fig 2 Amount of conjugated m-methybenzoic acid excreted 1.0
c , g
E 5 0.s
, a; x L
C
Fig 2 Amount of conjugated m-methylbenzoic acid excreted
by the individuals during 8-h exposure (black symbols) and oduring 24-h of collecting urine (empty symbols) and the averages of the groups during 8 h (hatched bottom part of the columns) and during 24 h (whole columns) without (C) and with (P) phenobarbital pretreatment
P
ment Figure 2 shows the amount of MBA excreted during 8 h of exposure and during 24 h from the beginning of the exposure; symbols corresponding to each individual and mean values for the whole group are given It is evident that phenobarbital pretreatment had no effect on the amount of excreted m-xylene metabolite. Excretion of M-methylbenzoic Acid (MBA) in Rats The amount of MBA excreted by rats is given in Figure 3 as the arithmetic mean of the values calculated per rat The exposure to concentrations of 400 and 800 mg m-xylene/m 3 air, respectively, gave similar results as in humans, i e , the difference between the amounts excreted by control and by phenobarbital pretreated rats was not statistically significant The twofold rise in the concentra-
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121
mg 90 80 Z 70 o
u 60 O 50
Fig 3 Arithmetic means of the amount of m-methylbenzoic acid calculated per rat, excreted during 6 h of exposure (hatched bottom part of columns) and during 24 h of collecting urine (whole columns) Vertical bars indicate 95% confidence intervals for means Asterisks indicate statistically significant differences between values before (C) and after (P) phenobarbital pretreatment (P< 005)
40
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Fig 4 Amount of m-methylbenzoic acid (Y) excreted per rat during 6 h of inhalation as a function of m-xylene concentration in inhaled air (X) Experimental points are the mean values for untreated (C-empty circles) and pretreated (P-black dots) rats
tion of xylene vapors (from 400 to 800 mg/m 3 ) doubled the amount of the excreted metabolite. However, a further increase in the concentration of xylene vapors (2000 and 4000 mg/m3 ) revealed a striking difference The control rats excreted during the exposure a similar amount of the metabolite regardless of the rising concentration of xylene in the air; merely the amount of MBA excreted during 24 h increased The increase, however, was not proportional to the rise of xylene concentration in the air (if it is assumed that the retained amount should be proportional to the product of the concentration and the duration of exposure).
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20 40 g/m 3 m-xylene O4 08 Fig 5 Mean amount of m-methylbenzoic acid excreted by rats during 6 h of exposure, expressed as the percent of the total amount excreted during 24 h (= 100 %) Denoted as in the foregoing figures
On the other hand, the amount of MBA excreted by phenobarbital pretreated rats increased during the exposure as well as during 24 h The excreted amount in this group corresponded well to the exponential course according to the equation y= 53 ( 1-e-0 35 X), where x is the concentration of xylene in g/m 3 and y is the
amount of MBA excreted per rat during 6 h of exposure in mg The corresponding equation for untreated rats is y = 13 (1-e-l' 4 x), but the agreement with the measured values is not as good (Fig 4). Figure 5 expresses the amount of MBA excreted during 6 h of exposure as the per cent of the total amount excreted during 24 h A statistical analysis confirmed the significance of the difference between the control and pretreated animals at the concentrations of 2000 and 4000 mg/m 3; moreover there was also a significant difference from the values obtained at 800 mg/m 3.
Discussion The study was primarily designed to investigate a possible impact of changes in the organism's metabolic activity on results of biological exposure tests, used for evaluating the level of industrial exposure to toxic chemical substances and based on the determination of corresponding metabolites of the substances in urine. When examining subjects exposed under entirely identical conditions, a certain variability of results is always found This can be seen also in our small human group (see Fig 2) As the cause of the variability, differences in the extent of lung ventilation and in the degree of the retention and metabolism of the substance are considered It is, therefore, of importance to know the factors that are responsible for the degree of retention-whether they are predominantly physico-chemical
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parameters of the retention compartment of the body or the biological component, i e , a different ability to metabolize xenobiotics. Xylene is suitable for such a study because it is metabolized by microsomal enzymes in the liver almost exclusively to a single metabolite-conjugated mmethylbenzoic acid, which is not physiologically present in urine. Phenobarbital, the most widely used experimental inducer with a broad spectrum, served for inducing enzyme activity Phenobarbital is also a frequent constituent of various drugs (hypnotics and analgesics) This was just the required situation: to see whether occasional therapy with drugs capable of enzyme induction affects the amount of retained xylene and/or the excreted metabolite. At the same time phenobarbital induction represented a model of physiologically high enzyme activity or of increased activity induced by industrial chemicals. In our human experiments, doses of phenobarbital were used having a tolerable hypnotic effect (though unpleasant and making, e g , car driving impossible) and known to induce enzyme activity even after some days of administration (Conney, 1967 ; Cuccinell et al , 1965 ; Vesell and Page, 1969) We have also proved this effect in three subjects by measuring antipyrine half-life The effect corresponded to data in the literature (Vesell and Page, 1969) The second exposure to xylene was repeated 6 weeks after cessation of phenobarbital administration, i e , after a sufficiently long period for the increased activity to return to normal level (MacDonald et al , 1969). Phenobarbital pretreatment of humans affected neither m-xylene retention in lungs nor the amount of the excreted metabolite in urine Such was the case in animals exposed to similar concentrations However, in rats exposed to severalfold higher xylene concentrations the amount of excreted metabolite (MBA) was markedly increased by phenobarbital pretreatment Lack of inducing effect at exposure levels comparable to the human exposure indicates that under such conditions even the biotransformation capacity of the control rat's liver is sufficient for complete metabolism of inflowing xylene (extraction ratio close to 1) Any increase in xenobiotic metabolizing capacity appears therefore superfluous at this concentration level The limiting factor in the total balance of the xenobiotic metabolism is the concentration of xylene in blood, i e , the amount of xylene entering the liver. The situation differs if the supplied amount of xylene exceeds the physiological metabolic capacity of the liver In such a case, this capacity becomes the limiting factor That is probably why at high xylene concentrations in the air, the amount of the metabolite excreted during the exposure by control animals (Fig. 3, concentrations 2 and 4 g/m3 ) was not higher than at lower concentrations. However, the total amount of MBA excreted during 24 h was somewhat enhanced Apparently at a high concentration level, more xylene is retained in the whole organism The desaturation phase after termination of exposure is thus longer and xylene is available for biotransformation for a longer period of time and in a larger quantity. If the biotransformation capacity of the liver is increased by microsomal enzyme induction it appears capable of metabolizing quantitatively the supplied xenobiotic even at high xylene concentrations The exponential relations between m-xylene concentration in the air and MBA excreted indicate that the maximum
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amount of MBA one animal was able to form within 6 h of exposure increased that at higher xylene from 13 mg to 53 mg due to the induction They also indicate excreted during metabolite the of amount the concentrations the increase in be reached) would value asymptotic an of % 90 , e (i exposure would virtually stop 3 rats Thus, 3 pretreated in g/m 6 6 at and at 1 6 g xylene/m air in untreated the given under capacity metabolic in increase according to these results the quantitative This fourfold about is induction conditions of phenobarbital microsomal relation need not apply to other conditions, as induction of hepatic on a dependent markedly quantitatively and enzyme activities are qualitively (including variety of factors, which include the nature of the inducing agents differences. sex dose levels) and species and lead to enhanced The present study found that enzymatic induction does not the inspired air in concentration its unless metabolism of the absorbed xylene that this important is it practice, health exceeds a certain limit For occupational in the xylene for MAC accepted generally limit lies for man and rat above the 3 as mg/m 800 around apparently is rats working atmosphere The limit for excreted of cent (per evaluation of method one indicated by the observation that the mean values, MBA-Fig 5) showed statistically significant differences of In some significance no revealed values whereas tests of differences in absolute German 3), mg/m 650 (approx ppm 150 countries MAC are near this value: Japan 3 in Czechoslovakia the average concen), ppm (870 mg/m Federal Republic 200 3 (less than 50 ppm), but the ceiling mg/m tration for the whole shift is only 200 may exceed the wholeconcentration in value up to which occasional deviations 3 Limits, 1977). Exposure (Occupational mg/m shift time weighted average is 1000 xylene were for liver human the If the biotransformation capacity of known) then not is course, of (which, liver proportional to the capacity of the rat 3 (nearly mg/m 800 of concentration to at a whole-shift xylene exposure up of excreted MBA ppm) the exposure tests based on monitoring the amount 200 concentrations higher At results correct (gedivec and Flek, 1976 b) would give in the concentration xylene the to less metabolite would arise than corresponds high spontaneous its or induction by air A hepatic metabolic activity enhanced also result as (and metabolized of level could probably increase also the amount the affect could difference this whether of retained) xylene The question remains onset of toxic clinical changes. amount Hence, there is no doubt that, e g , attempts to calculate the absorbed acute in metabolite excreted the of an organic solvent from the amount of blood the cases such in because inhalation poisonings are burdened with errors derived amount is contains very high concentrations of the substances and the turnover of the whole the of balance decidedly lower than in reality (in the primarily biotransformation, without substance the percentage that is eliminated levels). by breath, increases at high dose been tested The human biotransformation capacity for m-xylene has not recent found have we However, either experimentally or in field measurements, air the in vapors styrene of concentration information about the relation of high the From 1976) , al et m 6 (Engstr urine in to the concentrations of mandelic acid a styrene concentration figures reproduced by these authors it appears that up to 3 excretion of mandelic acid increases linearly, but this of 150 ppm, i e , 600 mg/m ,
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does not apply to higher concentrations Corresponding to 150 ppm styrene are approximately 3 g mandelic acid/g creatinine, to 300 ppm styrene only 5 g The authors do not explain this difference; in our opinion the limit of the biotransformation capacity of the liver could have taken part in it. Finally, it should be kept in mind that the quantitative relations in the
excretion of metabolites after gastrointestinal absorption of toxic substances may differ because in this case the whole absorbed amount enters the liver directly and the concentration of the substance in the portal vein surpasses the metabolic capacity of the liver These conditions, however, exceed the usual occupational health considerations.
References Brodie, B B , Axebrod, J , Soberman, R , Levy, B B : The estimation of antipyrine in biological material J Biol Chem 179, 25-29 (1949) Conney, A H : Pharmacological implications of microsomal enzyme induction Pharmacol Rev. 19, 317-366 (1967) Cuccinell, S A , Sansur, M , Burns, J J : Drug interactions in man Lowering effect of phenobarbital on plasma levels of bishydrocoumarin (Dicumarol) and diphenylhydantoin (Dilantin) Clin Pharmacol Ther 6, 420-429 (1965) Engstr6 m, K , Hark 6nen, H , Kalliokoski, P , Rantanen, J : Urinary mandelic acid concentration after occupational exposure to styrene and its use as a biological exposure test Scand J. Work Environ Health 2, 21-26 (1976) Flek, J , Sedivec, V : Determination of toxic substances and their metabolites in biological fluids by gas chromatography VII Toluric or Toluic acids in urine Collect Czech Chem. Commun 38, 1754-1759 (1973) Fouts, J R , Gut, I : Industrial and environmental xenobiotics In vitro versus in vivo biotransformation and toxicity Amsterdam, Oxford: Excerpta medica 1978 Frantik, E , Kratochvile, K : Inhalation exposure apparatus with total evaporation of micropump delivered liquids In: Adverse effects of environmental chemicals and psychotropic drugs, Vol 2, pp 321-324 Amsterdam: Elsevier 1976 Gillette, J R , Mitchell, J R : Concepts in biochemical pharmacology, Part 3 Berlin, Heidelberg, New York: Springer 1975 Mac Donald, M G , Robinson, D S , Sylvester, D , Jaffe, J J : The effects of phenobarbital, chloral betaine and glutethimide administration on warfarin plasma levels and hypoprothrombinemic responses in man Clin Pharmacol Ther 10, 80-84 (1969) Occupational exposure limits for airborne toxic substances Occupational safety and health series, No 37 Geneva: International Labour Office 1977 Sedivec, V , Flek, J : The absorption, metabolism and excretion of xylenes in man Int Arch. Occup Environ Health 37, 205 217 (1976 a) Sedivec, V , Flek, J : Exposure test for xylenes Int Arch Occup Environ Health 37, 219-232 (1976 b) Sedivec, V , Flek, J , MrAz, M : Preparation of an atmosphere with defined content of vapours of studied substances Prac L6k 26, 48-53 (1974) (in Czech) Vesell, E S , Page, J G : Genetic control of phenobarbital induced shortening of plasma antipyrine half-lives in man J Clin Invest 48, 2202-2209 (1969) Received January 29, 1979 / Accepted March 28, 1979
