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Original articleScand J Work Environ Health 1975;1(2):104-108

doi:10.5271/sjweh.2856

Exposure to methylene chloride. III. Metabolism of 14C-labelledmethylene chloride in rat.by Carlsson A, Hultengren M

Key terms: 14C-labelled methylene chloride ; animal exposure;carbon monoxide; exposure; metabolism; methylene chloride;radioactive atom; rat; tissue concentration

This article in PubMed: www.ncbi.nlm.nih.gov/pubmed/1226505

Scand. j. work environ. & heaLth 1 (1975) 104-108

Exposure to methylene chloride

ill. Metabolism of He-labelled methylenechloride in rat

by ANDERS CARLSSON, M.B., and MAY HULTENGREN, engineer 1

CARLSSON, A. and HULTENGREN, M. Exposure to methylene chloride: III. Metabo­lism of 14C-labeled methylene chloride in rat. Scand. j. work environ. & heaLth 1(1975) 104-108. The accumulation of methylene chloride and its metabolites in dif­ferent organs and tissues was studied in an experimental series comprising 10 rats.Each rat was exposed for 1 h to radioactive methylene chloride (14C) in a concentra­tion amounting to 1,935 mg/m3 in inspiratory air. Radioactive carbon atoms werefound in the isolated carbon monoxide after exposure. There was also a close cor­relation between the activity of carbon monoxide extracted from the blood sampleand the amount of carboxyhemoglobin (COHb) in the specimen. The largest concen­tration of methylene chloride and its metabolites per gram of tissue was found inwhite adipose tissue. This concentration had declined by more than 90 % 2 h afterexposure, whereas the concentration in the liver declined by about 25 % during thesame period. The amount accumulated in the brain displayed a decline of about 75 0/0

2 h after exposure. Thus the examination showed that the increased concentrationof COHb in the blood during exposure to methylene chloride is due to the metabolismof methylene chloride into carbon monoxide.

Key words: methylene chloride, animal exposure, metabolism, radioactive atoms,carbon monoxide, tissue concentration.

The inhalation of methylene chloride(CH2C12) has been found to result in theformation of carbon monoxide in man (I,4, 7). Endogenous carbon monoxide isnormally formed in the human body dur­ing the breakdown of hemoglobin (5, 8).An increase in ,this breakdown or someother change during exposure to meth­ylene chloI'ide may be an explanation forthe rise in carboxyhemoglobin (COHb)levels (3). Ano1Jher possibility is that themethylene chloride taken up by the bodyis rnetaJboliz,ed into, e.g., oa,rbon monoxide.

Work Physiology and Techonology Divisions,Department of Occupational Medicine, Na­tional Board of Occupational Safety andHealth, Stockholm, Sweden.

Reprint requests to: Mr. Anders Carlsson,Kungl. Arbetarskyddsstyrelsen, Arbetsmedi­cinska avdelningen, Fack, 100 26 Stockholm 34,Sweden.

104

These issues and the accumulahon ofmethylene chloride and its metabolites incer1Jain organs and tissues of the ra:t werestudied in the present investigation.

EXPERIMENTAL DESIGN

Ten male rats (Sprague-Dawley), eachweighing about 200 g, were used for theexperiment (Anti-cimex, Sollentuna, Swe­den).

The air mixture used in exposure wasprepared by injecting, with a microlitersyringe (Hamilton-Boyd, Switzerland), agiven quantity of radioa,etive methylenechloride (New England Nuclear, Mass.,U.S.A.) into a bag (specially made of poly­ester-laminated aluminium fOll) containinga known quantity of air. The radioactivemethylene chloride had a specific activityof 0.45 mCi/mmol.

The rats were placed in a cage (150 mminner diameter, Kebo-Grave, Sweden),which was well-.sealed. The air mixtureflowed from the aforementioned bag intothe cage via a rotameter (model 1350 V,Max Sievert, Vallingby, Sweden). Thesurplus air mixture was removed througha tube to which a water suction unit wasconnected. The entire device w.as placedin a fume cupboard. The rate of flow,which was ,adjusted with a tube clip, wasset ,at 20 l/h. Pr,etrials showed that thisrate supplied the rat with adequate quan­tities of oxygen and guaranteed a constantoage temperature of aboUJt 23°C.

After 1 h of exposure to about 1,935 mg/m 3 of methylene chloride in the inspira­tory air the rats were killed by means ofluxation of the cervical spine. The thor,axwas then opened by direct heart puncture,and blood samples were taken whichvaried from 0.5 to 1.6 m1. Approximatelyequal amounts of this blood were thentransferred to two injection bottles. Sam­ples were also ta'ken for determininghemoglobin concentration (cyanmethemo­globin method with spectrophotometricreading). Specim~ms were taken from theliver, kidneys, adrenals, white adiposetissue, cerebrum, and cerebellum. Thesespecimens were then assayed for theirradioactive content of methylene chlorideand metabolites.

ANALYTICAL METHODS

The concentration of methylene chloridein inspir.atory ,air was ootermin:ed witha gas chromatograph (1), and it proved tohave a mean value of 1,935 mg/rn3 (SD =± 90 mg/m3).

Blood carbon monoxide is normallybound to hemoglobin in the form of COHb.When the COHb content of blood was ana­lyzed, the ,released gaseous carbon mon­oxic1e was mea,sured. Radiooctive meth­ylene ch10ride was also rele-ased from theblood. A:J)ter 'the g,as (8 ml) had been col­lected in a 10-ml all-glass syringe (Becton,Dick-inson and Co., U.S.A.), it passed fivetimes over activated charcoal looselypaclmd in a 2-,cm long latex tube with ,aninner diameter of 0.3 cm (fig. 1). The meth­ylene chloride was absorbed, whereas thecarbon monoxide passed over. The meth-

Analysis ofCHp,I Analysis of CO

IX:'", .~. [ l~~o-_- c ~O'/.~ (----.

¥--_. ¥---D E -.:.',- ,

Liquid scintillation counting

Fig. 1. Method for measuring the radioactivecontent of formed carbon monoxide: The gasfrom the blood sample consisting of carbonmonoxide and methylene chloride was collectedin an all-glass syringe (A). This gas mixturepassed over activated charcoal (B) when themethylene chloride was absorbed. After pas­sage over the activated charcoal the gas wasanalyzed for methylene chloride and carbonmonoxide. Thereafter, the gas was allowed topass over hopcalite (C), a substance whichcatalytically converts carbon monoxide intocarbon dioxide. Another analysis was madeof the carbon monoxide content of the gas.In the first method the formed carbon dioxidewas dissolved in 3 ml of Carbosorb (D), intowhich scintillation liquid was mixed, and theradioactivity of the sample was measured.In the second method the carbon dioxide wasallowed to bubble through 3 ml of 0.02 M cal­ciumhydroxide. The precipitate obtained wasremoved. To this precipitate scintillation liquidwas added and the radioactivity of the samplewas measured.

yle.ne chLoride content of the gas was ana­lyzed after passage ov,er the activatedcharcoal so as to check on the degree ofabsorption. This check was made with ag,as chromwtogr:aph. After passing overthe activated aha'rcoal, the gas was checkedfor carbon monoxide content with a gaschromatograph. The error of the methodfo,r a single determinartion, calculated for10 double determinations, was 3.0 % of themean value of 4.25 % COHb (1).

In ord€r to facilitate simple measure­ment of the radioactive content of thecarbon monoX!ide, the carbon monoxidewas converted into carbon dioxide, whichis soluble in ,a liquid '8cintHlation sySitem.Thus the gas was allowed to paS's fivetimes over hopcalite (40 % CuO +60 Ofo Mn,p) , a substance which catalyti­cally converts carbon monoxide into car­bon dioxide. The hopcalite was al'8o loose­ly packed in a latex tube. An analysis ofthe carbon monoxide content of the gas

105

20

40

A.E.S.

The radioactive content of the carbonmonoxide formed in exposure closelyoorrelated with the amount of COHbformed. As previously mentioned, doubleblood samples were taken after everyexposure. These samples were processedin different ways aFter the absorption ofmethylene chloride and the conversion ofcarbon monoxide into carbon dioxide.

The first method, in which carbon di­oxide wa!S dissolved in CalI'hosOiTb, pro­duced a close correlation between theamount of COHb formed and the radio­activ.e content of carbon monoxide (fig. 3)(correlation coefiiicient r = +0.893). Acorrection was made for the ralt's COHbcontent prior to exposure. The mean valuefor the four controll raits was used (0.26 0/0;SD = ± 0.02 0/0).

The second method, in which the carbondioxide was bubbled through Erne water,resulted in a pr,ecipitation of calcium car­bonate (6). Close correlation was also foundhere between the activity in the precipita~e

and the amount of COHb formed (correla­tion coefficient r = + 0.936). Slnce theparticles of calcium carbonate fOI'Illed didnot remain suspended in the scintillationliquid, but were allowed ·to settle, thesecond method did not result in a countas high as the first method.

RESULTS

Fig. 2. Calibration curve for measuring abso­lute activity. The horizontal axis stands forthe external standard channels ratio (A.E.S.)and the vertical axis stands for the countingefficiency as a percentage of 14C (rj).

Radioactive measurement of carbonmonoxide

60

80

was made after it passed over the hopcalitein order to check on the conv'ersion of car­bon monoxide into carbon dioxide (fig. 1).

The carbon dioxide obtained in tiillsmanner was dissolved in 3 ml of Carbosorb(Packard InstrUlment AB, Bandhagen,Sweden), into which 12 ml of scintillationliquid, P,eI'lIIlaffiuor V (Packard Instr. AB),was mixed.

An additional blood sample was ta:kenfrom each Dat. This sample was processedin the sam€ manner as the first, with theexception that th€ carbon dioxide formedwas allowed to bubble through 0.02 Mcaldum hydroxide (lime water) (fig. 1).The precipitate obtained was remorved andmixed with 10 ml of scintillation liquidconsis·ting of 5.5 g of PeDmablend III (Pac­kard Instr. AB) dissolv·ed in 1 I of toluene.

The specimens taken from oI1gans andtissues were weighed and then dig€Stedfor 2-4 h at +50°C in 1 ml of Soluene-350(Packard Instr. AB). Nine milliliters ofthe Permab1end III scintillation liquid dis­solved in toluene was then added.

A liqUlid scintillation counter was used(mod.el 2425 Tri Carb, Packard Instr. Co.Inc., Downer Grov·e, III., U.S.A.). A 100 0/0counting efficiency is not usually .atltainedwhen the activity of soft beta particlessuch as 14C is measured. There are manyreasons for this occurrence, one being dis­coloration and another being the presenceof substances whioh impede the emissionof light from the solute. Such occurrencesare oalled quenching. A series of sampl€swere analyzed with the same known levelof activity (14C toluene), but with differ­ent quenching fa·ctors. The calibrationcurve (fig. 2) obtained in this manner madeit possible tooonvert a specimen's COUlllt­per-11Ilinute value into absolute aotivity,i.,e., disintegl1ation per minute (dpm). Astanda'rd sample with a known level ofactivity (HC-toluene) W2.tS also used ateach measurement in order to oheck thestabiHty of the scint1l1atiJon counter. Back­ground counts for each liquid scintJ!Jlationsystem were aliSO measured. PolyethylenescintililClition vials (Rackard Instr. AB) wereemployed in the experiments. Each spec­imen was measUI"ed in two cycles of either20 min or a maximum of 20,000 Lmpulses.The value for aotivi<ty was calculated asthe mean value of these two determin·a­tions (SD = ±0.7 Ofo ,in 20,000 impulses).

106

200

dprn x 103

•

•

Kubic et al. (2) showed that carbonmonoxide is forIlled after an intraperi­toneal injection of methylene ohloride intorats and that this carbon monoxide is ametabolite of methylene chloride. In thepresent study rats were exposed to 14C_labelled methylene chloride in the inspira­tory air. The increased level of carbonmonoxide proved to be derhned f.rommethylene chloride. Thus the methylenechloride taken up by the rat was metab­o.lized into products such ,as ·ca,r:bon mon­oxide.

Stewart et al. (7) showed that no increasein the level of urobilinog.en could bedemonstrated in man exposed to meth­ylene chloride. Thus there was no in­creased endogenous formation of carbonmonoxide due to an enhanced meta,bolismof hemoglobin. There should be no doubtthat the increased amount of carbonmonoxide formed during expo.sure tomethylene ohLoride is a metabolite of themethylene ohloride taken up.

Carbon monoxide is dangerous becauseit impedes oxygen transport. The half­life of carbon monoxide is prolong.ed dur­ing exposure to methylene chloride a,scompared to ordinary exposure to carbonmonoxide. This occurrence was pr,eviouslypointed out by Stewart et al. (7), and it hasbeen comfirmed in the present study.

COHb 9 x10-3

4.0 5.0 6.03.0

•

2.0to

•

•••

IFig. 3. Absolute activity (dpm. 103) in relationto the amount of carboxyhemoglobin (COHbg . 10-3). Every point stands for one exposedrat.

•

50

100

150

Accumulation of methylene chloride andmetabolites in organs and tissues

• liver• kidneys... adrenals... white adipose tissue

• brain

White adipose tissue contained the highestconcentration per gram of tissue imme­diatlely ,after the termination of exposure(fig. 4). The concentration of methylenechloride and metabolites in this tissue andin the brain diminished very rapidly.Levels in the liver, kidneys, and adrenalsdeclined far more slowly during the 6-hfollow-up after exposure.

DISCUSSION

75

50

25 \.....~ •...

-.

2 3 4 5

-'':::::----.t. time after

-- _=:'f exposure

6 hours

The results show that the concentration ofCOHb in blood increases -during exposureto methylene chloride in inspiratory air.This circumstance has been noted in anumber of studies (1, 4, 7).

Fig. 4. Accumulation of methylene chloride(p:g CH2C12/g tissue) and its metabolites inorgans and tissues during the 6-h follow-upafter exposure to methylene chloride in theinspiratory air.

107

REFERENCES

1. ASTRAND, 1., OVRUM, P. and CARLSSON,A. Exposure to methylene chloride: 1. Itsconcentration in alveolar air and bloodduring rest and exercise and its metabolism.Scand. j. work environ. & health 1 (1975)78-94.

2. KUBIC, V. L., ANDERS, M. W., ENGEL,R R, BARLOW, C. H. and CAUGHEY,W. S. Metabolism of dihalomethanes tocarbon monoxide. Drug metab. disposition2 (1974) 53-57.

3. NUNES, A. C. and SCHOENBORN, B. P.Dichloromethane and myoglobin function.MoL pharmacoL 9 (1973) 835-839.

Received for publication: 1975-02-10

108

4. RATNEY, R S., WEGMAN, D. H. and EL­KINS, H. B. In vivo conversion of methylenechloride to carbon monoxide. Arch. environ.health 28 (1974) 223-226.

5. SJOSTRAND, T. Endogenous formation ofcarbon monoxide in man. Nature 164 (1949)580.

6. SNEED, M. C. and MAYNARD, J. L. Gener­al inorganic chemistry. Chapman and HallLtd., London 1943, p. 643.

7. STEWART, R D., FISHER, T. N., HOSKO,M. J., PETERSON, J. E., BARETTA, E. D.and DODD, H. C. Carboxyhemoglobin eleva­tion after exposure to dichloromethane.Science 176 (1972) 295-296.

8. WHITE, P. Carbon monoxide productionand heme catabolism. Ann. n.y. acado sci.174 (1970) 23-31.