Pharmacokinetics of isoprene in mice and rats

9

Toxicology Letters, 36 (1987) 9-14 Elsevier

TXL 01735

PHARMACOKINETICS

OF ISOPRENE

(Isoprene; rats; mice; inhalation;

H.

PETERa,

J.G.

H.J.

WIEGANDa,

IN MICE

AND RATS

pharmacokinetics)

H.M.

BOLTa,

H.

GREIMb,

G.

WALTERb,

M.

BERGb

and

FILSERb*

Toxikologie und “Institut fiir Arbeitsphysiologie an der Universitiit Dortmund, Abteilung Arbeitsmedizin, Ardeystrasse 67, D-4600 Dortmund I, and bCesellschaft fiir Strahlen- und Umweltforschung, Institui fiir Toxikologie, Ingolstiidter Landstrasse 1, D-8042 Neuherberg (F.R.G.) (Received

8 September

(Revision

received

(Accepted

1986)

27 October

29 October

1986)

1986)

SUMMARY Pharmacokinetic saturation

analysis

kinetics

rate of metabolism

is directly

the body at low atmospheric amounts

of isoprene

in both species.

of isoprene

taken

inhaled

by male Wistar

Below atmospheric

proportional

to the concentration.

concentrations

suggests

up are exhaled

transport

as unchanged

values

further

of 130 pmol/(h

400 amol/(h

in proportion x kg) body

Isoprene

is

1.9 pmol/(h

mono-epoxides

endogenously

and systemically

and

substance

correspondence

Abbreviations:

0378-4274/87/$

FID,

03.50

flame ionization

0

Elsevier

is

detector;

Science

(15% in rats and 25% in mice). Its

concentration.

It finally approaches above

should

available.

and

limited produc-

Its

production

Part of the endogenous

extent: the rate of metabolism

be addressed.

B.V. (Biomedical

Division)

rate

is

isoprene

of endogenously

x kg) (rats) and 0.3 pmol/(h

CC, gas chromatography.

Publishers

maximal

1500 ppm in rats,

at high concentrations.

is 1.6 pmol/(h

requests

in

Only small

concentrations

systemically

to a greater

isoprene

and reprint

of isoprene

of the metabolsm.

above 300 ppm the rate of metabolism

x kg) in mice, respectively.

but it is metabolized available

The low accumulation

above 2000 ppm in mice. This indicates

of isoprene

produced

x kg) in rats, and 0.4 pmol/(h

is exhaled by the animals

*To whom

at atmospheric

x kg) body weight at concentrations

tion of the two possible

produced

to the atmospheric

weight

mice showed

of 300 ppm in rats and in mice the

limitation

half life in rats is 6.8 min and in mice 4.4 min. At concentrations does not increase

rats and male B6C3Fl

concentrations

x kg) (mice).

IO

INTRODUCTION

Isoprene, 2-methyl-1,3-butadiene is a structural analogue of chloroprene (2-~hloro-1,3-butadiene) and is mainly used for the synthesis of elastomers. Longo et al. [l] have shown that liver microsomes of various rodents including rats and mice metabolize this chemical to its two corresponding epoxides 3,4-epoxy3-methyl-1-butene and 3,4-epoxy-2-methyl-1-butene (20% in mice and 25% in rats). The latter is further oxidized by liver microsomes of all rodents tested to 2-methyl-1,2,3,4_diepoxybutane which, in contrast to the mono-epoxides, is mutagenic in ~fff~o~eff~ ~~~~i~u~fu~ 121.From these results the authors concluded that isoprene may be a potential carcinogen, although it was not mutagenic in bacterial test systems using rat-liver microsomes [3]. To evaluate the possible carcinogenic risk of isoprene, information on the distribution and metabolism in the intact animal is required. Therfore, the pharmacokinetics of isoprene in male BK3Fl mice and male Wistar rats have been investigated. MATERIALS

AND

METHODS

Male Wistar rats (200-250 g body weight) and male B6C3Fl mice (25-30 g body weight) were purchased from Versuchstierzucht, Hannover (F.R.G.). lsoprene, 99% pure (gold label), was obtained from Aldrich, Steinheim (F.R.G.). In every experiment 2 male Wistar rats or 5 male B6C3Fl mice were exposed to gaseous isoprene in closed exposure systems (volume 6.4 1) as previously described [4,51. Initial concentrations of isoprene ranging from 5-1000 ppm were adjusted by injecting the gaseous mixtures with air. Higher concentrations of isoprene, up to 4000 ppm in the gas phase, were generated by direct injection of the hightly volatile liquid into the system. The concentration in the exposure system was measured by GC. We further determined the Ostwald partition coefficients for isoprene between olive oil and air and between saline and air at 37°C using a head space method as discribed in [6]. The conditions of gas chromatography were 300 ml/min air and 30 mlimin hydrogen for FID, detector temperature 2OO”C, l/8 inch glass column, 2 mm i.d., length 3 m, filled with Tenax GC, 35-60 mesh (Latek, Heidelberg, F.R.G.), carrier gas nitrogen, flow 60 ml/min, column temperature 150°C. Chemical homogenecity of isoprene was checked with another l/8 inch stainless steel GC colmn, length 3 m, filled with Porapak Q, 60-80 mesh (Latek, Heidelberg, F.R.G.). The other conditions were as above. Each gas sample from the atmosphere in the exposure system was entered into a 2-ml gas-sample loop and analyzed by GC. Pharmacokinetic analysis was carried out using a two-compartment model as described earlier [5]. Isoprene is produced endogenously and exhaled by rats and man [7]. We estimated this by analyzing the exhaled air of non-exposed animals kept in the closed exposure system. Detection limit for isoprene by the Tenax GC column was 0.01 ppm.

11

RESULTS AND DISCUSSION

Each concentration-decay curve shown in Fig. 1 represents an experiment with 2 male Wistar rats (Fig. 1) or 5 male B6C3Fl mice (Fig. l), respectively. The animals were exposed to different initial concentrations of gaseous isoprene, up to 4000 ppm, in our closed exposure system (for details see [4,5]). Time-dependent concentration decline in the atmosphere of the system was determined by GC (Fig. 1). Similarly, exhalation and accumulation of endogenously produced isoprene was determined in untreated mice and rats (see upward curves in Fig. 1). These data were analyzed to determine the pharmacokinetics of isoprene in rats and mice. In both species, metabolism of isoprene shows saturation kinetics (Fig. 2). Below concentrations of about 300 ppm, metabolism is almost directly proportional to the atmospheric concentration of the substance. However, even in the absence of exogenous isoprene, substantial amounts are metabolized, since isoprene is produced endogenously (see upward curves in Fig. 1). The rate of metabolism of systemically available endogenous isoprene is calculated to be 1.6 pmol/(h x kg) in the rat and 0.31 pmol/(h x kg) in the mouse.

1o-23

9 IO t,me [h]

Fig. 1. Concentration-time by 2 rats graphical

and

5 mice,

extrapolation.

10-2

I , , , , , , , , , , , 0

12

curves of isoprene in closed exposure

respectively,

in each

experiment.

Open

3

L

systems circles,

5

6

7

of 6.4.liter measured

8

9

10

volume values;

tome [h]

occupied

solid lines,

coneIppml

0 0

1000

2000

Fig. 2. Rate of metabolism concentration.

3000 (dN,t/dt)

Dots, calculated

lines, graphical

of isoprene

in rats (left) and mice (right) dependent

rates of metabolism

(1 kg body weight;

open exposure

on atmospheric system);

dotted

extrapolations.

The maximal velocity (I’,,,) for metabolism of isoprene in mice [400 pmol/(h x kg)] was 3 times higher than in rats [ 130 pmol/(h x kg)]. This species difference has also been reported by Logo et al. 1985 [l]. In incubations with liver microsomes from Albino Swiss mice V,,, was about 7 times higher than in incubations with microsomes from Wistar rats. TABLE

I

PHARMACOKINETIC

PARAMETERS

Parameter partition

body/air);

Concentration (whole Clearance

(related

concentration Vlklz= Clearance

from

tissue

ppm in atmosphere

concentration

(related

* 2000

16000

+ 3000

ml/h

1000

12000

t

3000

ml/h

940 * 300

2 300 * 1 000

ml/h

6.8 +- 2.4 130 (see Fig. 2)

4.4 + 1.5 400 (see Fig. 2)

1.9 + 0.8

0.4 * 0.2

to

in the atmosphere);

VzKstke? Clearance of exhalation

6200+ (related

in the body);

lifeb; ln2/(k,t

to the Vzkzla

+ k21ja

Maximal rate of metabolism; (V,,,,,)” Endogenous production ratec; dNpr/dta Rate of metabolism

0.3 * 0.2

1.6 + 0.7

for: 1 kg body weight ( VZ = 1000 ml); dynamic constants

calculated

according

(open) exposure

to the two-compartment

‘Between 50 and 250 ppm (rat) and between 10 and 300 ppm (mouse). ‘Calculated for the systemically available isoprene. f

min amol/h/kg pmol/h/kg

of endogenously

isopreneC

to ]4,51). aPharmacokinetic

dMean value

nl gas/ml

1.7 + 0.6

1.2 -t 0.4 the

7300

produced

tissue

ppm in atmosphere

to the

of metabolismb

Calculations

nl gas/ml

7.0 * 2

in the atmosphere);

the concentration

Half

3

stateb

KS,”

of uptake

atmosphere

7.8 f

in steady

body/air);

Dimension

coefficient

Ke,a

ratio

IN RATS AND MICE Miced

Rat?

Thermodynamic (whole

OF ISOPRENE

S.D. of three exposures

with 2 rats,

5 mice, each.

pmol/h/kg system (VI +y model

[4,5].

according

13

In Table I the p~armacokinetic parameters for isoprene in rats and mice are summarized. Accumulation of isoprene in the organism is determined by the rates of uptake via inhalation, exhalation and metabolism. Isoprene accumulates in the organism as long as rates of inhalation exceed rates of exhalation and metabolism. At high concentrations, when metabolizing enzymes are saturated, accumulation is determined only by the rates of inhalation and exhalation, whereas metabolism becomes negligible. At such conditions accumulation is determined by the thermodynamic partition coefficient which represents the concentration equilibrium between the organism and the atmosphere [4]. Here, accumulation is very similar in both species investigated: 7.8 times in rats and 7.0 times in mice in relation to the solubility of isoprene in the tissues of the animals. This solubility can also be estimated as described in [6] by means of the Ostwald partition coefficients for olive oil/air, and saline/air, respectively. At 25°C these partition coefficients were determined to be 75 -+ 1.2 (nl gaseous isoprene per ml olive oil divided by ppm isoprene in atmosphere; n = 4), and to be 0.52 f 0.004 (nl gaseous isoprene per ml saline divided by ppm isoprene in atmosphere; n = 4). Assuming for an animal a lipid content of 10% and a water content of 70%, while the rest of the organism is neglected, and setting solubility in olive oil and in saline equal to that in lipid tissue and in body water, respectively, we estimate the thermodynamic partition coefficient whole body/atmosphere for isoprene to be about 7.9. At atmospheric concentrations below 250 ppm (rat) and 300 ppm (mouse), concentration ratios between whole body and atmosphere are lower than could be expected from the partition coefficient. At such concentration ranges only limited accumulation is observed. In both species metabolic clearance is similar to the clearance of isoprene uptake from the gas phase. This indicates that most of the isoprene entering the body is metabolized. Only a minor portion is exhaled unchanged (15% in rats and 25% in mice as can be calculated by dividing the difference between the clearances of uptake and of metabolism by the clearance of uptake, multiplied by 100). Considering this and the limited accumulation of isoprene we conclude: At low concentrations the rate of metabolism of exogenous isoprene is likely to be limited by the transport to the metabolizing sites, rather than by metabolic capacity. The half life for isoprene inhaled from the atmosphere of an infinitely large volume has to be equal to the half life for isoprene leaving the body after the end of exposure as it is solely determined by the clearances of exhalation and metabolism [8]. At atmospheric concentrations below 300 ppm it is calculated to be 6.8 min in rats and 4.4 min in mice. As metabolism is saturated at high concentrations, clearance of metabolism decreases with increasing concentration. Therefore, at very high concentrations the half life is finally assessed by clearance by exhalation, only. It increases up to 44 min in rats, and to 18 min in mice (In 2/k2r according to [4,5]). Isoprene is produced endogenously as demonstrated in exhalation experiments with untreated animals (Fig. 1, table). This has also been observed in man [7]. The

14

production rate of endogenous isoprene in rats and mice compared with that of endogenous n-pentane or ethane [9] is 2 to 3 orders of magnitude higher. Considerations on a possible carcinogenic or mutagenic potential of isoprene metabolites should take into account the endogenous production of isoprene. For rats and mice, we have estimated the amounts of endogenous, systemically available isoprene which in a first metabolic step is biotransformed to the non-mutagenic monoepoxides. However, the exact metabolic pathway leading to the mutagenic diepoxide in living animals is still to be clarified. A subsequent risk estimate for exogenous and endogenous isoprene might then be performed as exemplified by ethylene and ethylene oxide [lo,1 11. ACKNOWLEDGEMENT

The authors thank Roman Koch for his help in editing the manuscript.

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