Effect of chloroform on the activities of liver enzymes in rats

Toxicology, 14 (1979) 23--38 © Elsevier/North-Holland Scientific Publishers Ltd.

E F F E C T OF CHLOROFORM ON THE ACTIVITIES OF LIVER ENZYMES IN RATS

W.K.L. G R O G E R

and T.F. G R E Y

Environmental Safety Division, Unilever Research, Colworth House, Sharnbrook, Bedford (Great Britain) (Received April 9th, 1979) (Revision received August 15th, 1979) (Accepted August 21st, 1979)

SUMMARY

Rats were dosed once, 5 times or 10 times with chloroform (0.5--50 mg/ kg) and the liver enzyme activities were determined. Chloroform induced much change in the 24 enzymes investigated but caused only minimal liver enlargement. The main enzymic changes were: stimulation of glycolysis and oxidative phosphorylation, increased breakdown of protein and nucleic acids, hexose phosphate shunt activity was reduced leading to a shortage of NADPH in the cell, adrenal medullary and cortical secretion were stimulated. These results show t h a t ingestion of chloroform even in small amounts causes distinct changes in liver cell metabolism as indicated by enzyme activities. Some of the changes are similar to, but others differ from, those seen with larger and anaesthetic doses. In most cases a transition between these 2 types of effects can be seen.

INTRODUCTION

Chloroform has long been known as a hepatotoxic agent; typical effects on liver cells are extensiye vacuolisation, disappearance of glycogen, fatty degeneration, swelling and necrosis, all starting in the centrilobular areas [1--16]. There is also often haemorrhaging into the parenchyma and infiltration of polymorphonuclear cells and monocytes [17,18]. Chloroform causes morphological damage to liver cells even in vitro [19--21]. Electronmicroscopic observations of parenchymal liver cells from chloroformpoisoned animals were carried out by Scholler [22,23], to reveal deposition of lipid droplets in the cytoplasm, partial destruction of the mitochondrial matrix, proliferation of smooth endoplasmic reticulum and swelling of the rough endoplasmic reticulum with detachment of ribosomes.

23

In view of the histological evidence for the hepatotoxicity of chloroform, it is expected t h a t administration of this compound adversely affects the biochemical response to liver function tests. Such studies have been reported [5,9,12,15,24--32]. Only Imray et al. [24] and Kirwan et al. [26] found no significant changes. However, these 2 groups tested only 1 enzyme: serum alanine aminotransferase. In both reports the negative results may have been due to the extremely short duration of exposure to chloroform. Thorpe et al. [15] likewise demonstrated that of all the serum enzyme activities which they measured in chloroform-treated horses, only alanine aminotransferase activity remained within the normal limits. However, Scholler [31] found that the activities of liver cell enzymes reach their maximum levels in the serum of man between the third and fourth day after exposure to chloroform, whereas the authors cited above had measured the serum alanine aminotransferase activities in their patients within the first 24 h. This could account for their failure to find significant anomalies. Although it is clearly impossible to compare these sets of data, it appears that changes in the activity of serum alanine aminotransferase are not a valid indication of chloroform-induced liver injury. The influence of chloroform on the activities of some subcellular liver enzymes has been described before [14--16,33--37], but the results were obtained using anaesthetic doses of chloroform. In our study, we have examined the effects of low doses of chloroform on the activities of 23 enzymes and cytochrome P-450 in 4 subceUular fractions from rat liver. METHODS Analar grade chloroform from British Drug Houses Ltd. was used. 18 groups of rats, 6 males and 6 females per group, were orally intubated with solutions of chloroform in groundnut oil to give dose-levels of 0, 0.5, 1.0, 5.0, 25.0 or 50.0 mg chloroform/kg body wt, 3 groups for each doselevel. 1 group at each dose-level was killed after 1 dose, 5 doses and 10 doses. The rats in the 5 and 10 dose groups were treated once daily for 5 or 10 days. The age of the rats was 8 weeks on the day of sacrifice. The activities of the following enzymes were examined:Mitochondria:

Glutamate dehydrogenase Succinate dehydrogenase Adenosine triphosphatase 3-Hydroxyacyl-CoA dehydrogenase Acetyl-CoA acetyltransferase Pyruvate kinase Amine oxidase L y s o s o m e s and p e r o x i s o m e s :

~-Glucuronidase

24

Dipeptidyl peptidase (Cathepsin C) Acid phosphatase Arylsulphatase Urate oxidase Microsomes :

Azobenzene reductase Aryl-4-monooxygenase O-Demethylase N-Demethylase (Aminopyrine) NADPH-Cytochrome reductase

Arylesterase Cytochrome P-450

Isocitrate dehydrogenase Glucose-6-phosphate dehydrogenase Alanine aminotransferase Tyrosine aminotransferase

Soluble phase: Lactate dehydrogenase

Colworth Wistar rats were used and they were housed in individual cages in air-conditioned rooms at a temperature of 21°C-+ 2°C and h u m i d i t y 55 +- 5%. The rats were fed on Spital No. 1 diet ad lib. and had free access to water; the composition of the diet is shown in Table I. After the single or final dose the animals were starved for 16 h, killed by cervical dislocation and exsanguinated immediately; the livers were excised, weighed and each was immersed in 10 ml ice-cold 0.25 M sucrose solution. The livers were cut into small pieces and homogenised in a Potter-Elvehjem homogeniser consisting of a plain glass tube and a Teflon pestle. The homogenates were subjected to differential centrifugation in a Griffin-Christ Omega 2 Preparative Ultracentrifuge using a 3 × 30 ml swing-out rotor. Cell fractionation was carried o u t by the m e t h o d described by Schneider and Hogeboom [38]. Fractions in the form of pellets were resuspended in 0.25 M sucrose and these suspensions as well as the supernatant from the last particulate (microsomal) fraction were frozen in acetone-dry ice mixtures and freeze-dried in a Leybold G 07 Freeze Drying Plant. The dried specimens were sealed and stored in the cold-room at -15°C until the TABLE I COMPOSITION OF THE RAT DIET (SPITAL No. 1) Ingredient

A m o u n t (%)

Maize Barley Copra Meal germ Wheat feed White fLsh Dried yeast Extracted groundnut Spray dried milk powder Edible crude palm oil

10.09 5.04 10.09 2.52 25.94 8.4 8.0 10.09 12.56 0.84 0.33 1.00 5.04

Wheat

Limestone

Salt Molasses

Manganesesulphate Copper sulphate Iron oxide Vitamins A, B and D complex

} 0.06

100.00

25

t~

Reduction of 2-oxoglutarate in the presence of NADH= and NH4 ÷ Oxidation of succinate in the presence of 2-(4-iodophenyl)-3-(4-nitrophenyl}5-phenyltetrazolium chloride Dephosphorylation of ATP to ADP using pyruvate kinase and lactate dehydrogenase as indicator enzymes Reduction of acetoacetyl-CoA in the presence of NADH 2 Degradation of acetoacetyl-CoA to acetyl-CoA Degradation of phosphoenolpyruvate to pyruvate and use of lactate dehydrogenase as indicator enzyme Breakdown of kynuramine Hydrolysis of phenolphthalein-~-glucuronide Breakdown of casein and spectrophotometric measurement of liberated tyrosine at 280 nm Hydrolysis of p-nitrophenylphosphate Hydrolysis of p-nitrocatechol sulphate Oxidation of uric acid to allantoin Reduction of neoprontosil Hydroxylation of aniline Hydrolysis of p-nitrophenetole Hydrolysis of amidopyrine Oxidation of NADPH 2 in the presence of cytochrome c Hydrolysis of ~-napthylacetate Measurement of light absorption of reduced cytochrome and its carbon monoxide complex Reduction of pyruvate in the presence of NADH 2 Oxidation of isocitrate in the presence of NADP Oxidation of glucose 6-phosphate in the presence of NADP Deamination of l-alanine and measuring pyruvate with lactate dehydrogenase Deamination of 1-tyrosine

Glutamate dehydrogenase Succinate dehydrogenase

Lactate dehydrogenase Isocitrate dehydrogenase Glucose-6-phosphate dehydrogenase Alanine aminotransferase Tyrosine aminotransferase

Acid phosphatase Arylsulphatase Urate oxidase Azobenzene reductase Aryl-4-monooxygenase O-Demethylase N-Demethylase (Aminopyrine) NADPH-Cytochrome reductase Arylesterase Cytochrome P-450

Amine cxidase ~-Giucuronidase Dipeptidyl peptidase (Cathepsin C)

3-Hydroxyacyl-CoA dehydrogenase Acetyl-CoA acetyltransferase Pyruvate kinase

Adenosine triphosphatase

Method

Enzyme

METHODS USED FOR ENZYMIC ACTIVITY MEASUREMENTS

TABLE II

57 58 59 60 61 62

47 48 49 50 51 52 53 54 55 56

44 45 46

41 42 43

40

39

Reference

enzyme assays could be carried out. Previous studies in this laboratory have shown that no measurable changes occur in the activities of enzymes stored for up t o 1 m o n t h under these conditions. The enzyme activities were determined with the aid of a LKB 8600 Reaction Rate Analyser, an Optica C F 4 R Recording S p e c t r o p h o t o m e t e r and a Unicam SP600 Spectrophotometer. All the methods used were modifications or adaptations of the standard published techniques and are listed in Table II. RESULTS

In this study the values for the results obtained had to be assessed in relation t o 3 factors: (1) repeated administration o f groundnut oil; (2) administration o f different levels of chloroform; (3) repeated administration of chloroform. T A B L E IH BODY WEIGHTS AND LIVER WEIGHTS OF RATS TREATED WITH CHLOROFORM IN G R O U N D N U T OIL Chloroform doselevel (mg/kg)

Males

Females

N u m b e r of doses

N u m b e r of doses

1

5

10

1

5

10

194 197 202 195 187 190

192 194 203 199 198 198

138 134 128 126 129 133

128 132 140 135 134 130

129 132 131 133 131 129

7.54 7.35 7.68 7.39 7.36 7.57

7.08 7.25 7.48 7.49 7.86 7.38

5.17 5.10 4.65 4.69 4.83 5.24

4.77 4.71 5.31 5.09 5.35 5.12

4.62 4.68 4.77 4.90 5.06 5.13

3.69 3.75 3.71 3.77 3.99 3.72

3.75 3.81 3.63 3.74 3.76 3.94

3.74 3.56 3.78 3.77 4.00 3.95

3.60 3.55 3.64 3.68 3.85 3.98

Body weigh~ (g) 0.0 0.5 1.0 5.0 25.0 50.0

182 183 178 179 183 186

Liver weights (g) 0.0 0.5 1.0 5.0 25.0 50.0

7.35 7.27 6.77 6.80 7.31 7.67

Relative liver weigh~ (g/l O0 g body wt) 0.0 0.5 1.0 5.0 25.0 50.0

4.02 3.97 3.83 3.80 3.99 4.11

3.89 3.73 3.77 3.78 3.96 3.99

27

The data were evaluated mathematically because the various effects sometimes cancelled each other out and sometimes enhanced each other. The statistical methods used were analysis of variance and regression analysis in an attempt to eliminate the contribution of repeated administration of groundnut oil and to separate the effects of different dose-levels of chloroform and repeated administration of chloroform.

Body weights and liver weights (Table III) There were no significant changes in the body weights of male or female rats but repeated administration seemed to give rise to some increases, more apparent in females than in males. In contrast, a single dose caused a slight decrease in body weights of females. Liver weight changes were similar in males and females but were significant in females only; single doses caused a reduction in weight while repeated administration caused an increase. Relative liver weights followed the same pattern but the effects were much weaker, in particular the single dose effect in females was absent.

Mitochondrial enzymes (Table IV) Glutamate dehydrogenase activity was significantly increased but the effect was not dose-related. Repeated administration had little additional effect although the increase was slightly reduced after 10 doses. Both male and female rats responded similarly, although the effect was greater in the females. Succinate dehydrogenase activity was increased by a single dose of T A B L E IV AVERAGE TREATED Chloroform doselevel (rag/

ACTIVITIES O F L I V E R M I T O C H O N D R I A L E N Z Y M E S WITH CHLOROFORM IN G R O U N D N U T OIL

OF RATS

Glu tamate dehydrogenase

Succinate dehydrogenase

Adenosine triphosphatase

3-HydroxyacyI-CoA dehydrogenase

N u m b e r of dc~es (U/kg body wt)

N u m b e r of dc~es (kU/kg body wt)

N u m b e r of doses (U/kg body wt)

Number of doses (U/kg body w t )

kg)

I

5

I0

I

5

10

I

5

10

1

5

10

M~es 00 0.5 1.0 5.0 25.0 50.0

95.3 115.1" 113.8" 109.4" 108.6" 95.7

92.6 108.1" 112.7" 113.2" 1 15.3" 113.7"**

92.1 105.9 104.2"* 104.5" 97.2 101.9

19.7 21.6 21.6 24.0* 22.0 21.0

31.0"* 29.6** 28.9** 29.9** 29.1"* 31.2"*

22.2** 21.8 20.3 20.3** 20.1"* 19.8

140.7 148.6 138.3 132.7 123.2" 128.7

138.5 120.7"* 120.4"* 128.0 145.1"* 148.1"*

137.7 132.2 131.7 129.9 143.9 151.4"*

292.3 327.9* 328.2* 310.7 280.4 258.4*

237.6** 202.8*** 189.6"** 212.1"** 193.5"** 199.6"**

2 2 2 . 8 *~ 2 2 4 . 5 *~ 203.8** 195.2"* 227.9"~ 217.2"*

Females 0.0 0.5 1.0 5.0 25.0 50.0

235.8 288.5* 277.4* 305.9* 310.1" 286.9*

272.2** 283.4 261.1 276.9 290.0 315.0"

275.2** 315.4 292.3 305.1" 310.5" 325.8*

55.0 68.4* 67.5* 73.6* 65.7* 59.3

78.4** 77.2 78.7 79.9 77.4** 90.2***

71.0"* 77.6 74.4 71.2 84.6*** 84.3***

268.4 282.0 333.9* 335.4* 365.3* 363.9*

267.9 278.2 278.3 273.5** 297.4*** 328.8*

258.4 314.6 310.1 369.4* 422.9* 415.7"

373.3 405.8 502.4* 503.7* 500.4* 495.8*

357.5 367.4 343.4"* 347.9** 415.2"** 460.4*

4 6 7 . 8 *~ 451.1 464.6 460.6 513.6 635.8*

*Significantly d i f f e r e n t from control group values (P = 0 . 0 5 ) . * * S i g n i f i c a n t l y different from 1-dose group values (P = 0 . 0 5 ) . * * * S i g n i f i c a n t l y different from the c o n t r o l group values and the 1-dose values (P = 0 . 0 5 ) .

28

chloroform; this effect was reduced by multiple administration, and in the males the effect disappeared altogether after 10 doses. Adenosine triphosphatase activity in male rats was unchanged, but in females the activity was increased after 1 dose and was further increased by repeated administration. 3-Hydroxyacyl-CoA dehydrogenase activity in males was unchanged after a single dose, but repeated administration caused a slight inhibition. In females, chloroform increased the a,;tivity but the effect was reduced after repeated administration. Acetyl-CoA acetyltransferase activity was unchanged in males; in females, activity was significantly increased after 10 doses. Pyruvate kinase activity was unchanged in males; in females, activity was significantly increased and multiple administration had no additional effect. Amine oxidase activity was significantly increased in males and females after a single dose; repeated administration caused a reduction of the effect, more noticeable in females after more than 5 doses.

L ysosomal and peroxisomal enzymes (Table V) ~-Glucuronidase activity was decreased in females only; multiple administration had no additional effect. Dipeptidyl peptidase activity was significantly increased in males, but only slightly in females. Multiple administration had no additional effect in male rats but caused further slight increases in females. Acid phosphatase activity was increased in both males and females; multiple administration had no additional effect. Aryl sulphatase activity was increased in both males and females; repeated administration

Acetyl-CoA aoetyltrans ferase

Pyruvate kinase

Amine oxidase

Number of doses {kU/kg body wt)

Number of doees (U/kg body wt)

Number of doses (U/kg body wt)

1

45.3 45.5 48.7 4 9 . 5* 49.5 45.6

79.8 79.8 80.8 81.1 83.6 78.2

5

10

52.8** 52.6** 56.7** 63.9*** 70.5*** 53.9**

126.0"* 130.0"* 91.8" 126.4"* 126.4 *m 129.1"*

54.9** 54.6** 54.4 54.6** 64.6** 49.5

81.4 80.5 80.7 102.6"** 105.8" i03.0"**

1

5

10

45.5 45.7 47.1 43.4 47.5 44.6

43.4 39.8*** 36.4*** 37.5*** 42.7** 40.6

41.6 41.9 40.8** 41.4 42.9 41.5

56.2 59.0 62.5* 67.4* 78.4* 73.2"

55.6 58.2 62.3* 63.1" 64.3*** 64.1"

54.1 60.7* 59.5* 64.5* 66.3"** 71.0"

1

5

10

682 786* 762* 765* 785* 722

787** 754 766 742 795 791

1490 1528 1723" 1947" 1807" 1857"

1622 1687"* 1604 1565"* 1657 1647

714 709 757 749 782* 774

1631 1651 1574 1587"* 1810 1866"

29

CO O

3694 3137" 3188 3164" 3491" 3612

3580 3328 3370* 3076* 3397 3464

1843 1721 1832 1896 2003 2272*

1830 1892 2051 1987 2498*** 2428*

2624 2760 2653 2665** 3418" 3405*

1918 1907"* 2180"* 2335*** 2324*** 2079

2431 2710" 2753* 3075* 3011"** 2987*

5.08 5.19 5.21 5.57 5.80* 5.85*

7.77 7.77 7.74 8.55* 8.99* 9.43*

1

5.60** 5.66** 5.56 5.69 5.63 6.16"

7.50 7.74 7.49 7.79** 8.62* 9.14"

5

N u m b e r of d o s e s (U/kg body wt)

5.29 5.54** 5.31 5.59 5.76* 6.00*

7.41 7.36** 7.96 8.45* 8.55* 8.79*

10

Acid phosphateme

* Significantly d i f f e r e n t f r o m contxol g r o u p values (P = 0 . 0 5 ) . ** Significantly d i f f e r e n t f r o m the 1-dose g r o u p v a l u e s (P = 0.05). * * * S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p v a l u e s a n d the 1-dose g r o u p v a l u e s (P = 0.05).

3954 3680** 3673 3559** 3859** 4004

2585 2764* 2870* 3296* 3362* 3102"

Females 0.0 0.5 1.0 5.0 25.0 50.0

3891 3640 3487 3518 3880 3972

3663 3688 3563 3491 3547 3562

3734 3628 3573 3757 3610 3806

10

Males 0.0 0.5 1.0 5.0 25.0 50.0

5

1

1

10

N u m b e r of doses (Ulkg body wt)

N u m b e r of doses (U/kg body wt)

5

Dlpeptidyl peptidase

13-Glucuronidase

Chloroform doselevel (ms/ kg)

5431 5559 6231" 7147" 7554* 6306*

6518 7000 7033* 8746* 7797* 7924*

1

5618 5695 5757 5924** 6570* 6686*

7145"* 6878 6799 6903** 9565*** 9546***

5

N u m b e r of d o s e s (U/kg body wt)

Arylsulphatase

5965 7544* 7295* 7815" 8216" 8224*

1

6326** 6848 7836*** 8144 8023*** 8336* 8469*** 8417" 8404* 9436* 8991"** 9249*

6833 7682* 7727*** 8364* 8472* 8603***

10

9751"* 9596 10390** 9816"* 10101 9589

6913"* 7666* 7797* 8065* 9516"** 8697*

5

N u m b e r o f doses (U/kg body wt)

Urate oxidase

8328** 8198 9110 8919 9319" 11178***

7574** 8630*** 8474** 9185"** 9545*** 8958*

10

A V E R A G E A C T I V I T I E S OF L I V E R L Y S O S O M A L A N D P E R O X I S O M A L E N Z Y M E S O F R A T S T R E A T E D W I T H C H L O R O F O R M IN G R O U N D N U T OIL

TABLE V

caused an additional increase in females, but less so in males. Urate oxidase activity was increased in males and females but repeated administration reduced this effect.

Microsomal enzymes (Table VI) Azobenzene reductase activity was increased in males and females and repeated administration caused an additional increase. The changes were significant in females but not in males receiving less than 10 doses. Aryl 4-monooxygenase activity was n o t significantly changed in males after a single dose; repeated administration caused a slight inhibition. In females, 1 dose increased the activity which was further stimulated by repeated administration. O-Demethylase activity was stimulated in both sexes after 1 dose. Repeated administration increased this effect in males but reduced it in females. N-Demethylase activity was inhibited in males and females and repeated administration caused a further inhibition; the effects were stronger in males than females. Arylesterase activity was increased in males and females. Repeated administration caused a slight additional increase in females but in males the l
Soluble Phase Enzymes (Table VII) Lactate dehydrogenase activity was increased after 1 dose in males and females although more strongly in females. Repeated administration caused an additional increase but more strongly in males. Isocitrate dehydrogenase activity responded to chloroform similarly to lactate dehydrogenase. Glucose-6-phosphate dehydrogenase activity reacted differently in males and females. In males, activity was increased after 1 dose but repeated administration caused a decrease. In females, 1 dose caused decreased activity which was further decreased by repeated administration. Alanine aminotransferase activity also responded differently in males and females. In males, 1 dose*had no effect but repeated administration caused a decrease in activity. In females, activity increased after a single dose but repeated administration reduced this effect and after 10 doses activity was similar to t h a t of the control group. Tyrosine aminotransferase activity was increased in both sexes after 1 dose; repeated administration caused an additional increase in males only. DISCUSSION

Hitherto, the investigations on the effects of chloroform on the activities

31

T A B L E VI AVERAGE Ac~rIVITIES OF LIVER MICROSOMAL ENZYMES OF RATS TREATED WITH CHLOROFORM IN G R O U N D N U T OIL Chloro" form

Azobenzene reductase

Aryl 4-monooxygenas¢

O-Demethylase

N-Demethvlase

doselevel

N u m b e r o f doses ( U / k g body w t )

Number

Number

Number

o f doses

o f dos e s ( U / k g body w t )

( m U / k g body w t )

o f do~es

(kU/kg body w t )

(mg/kg) 1

5

10

1

b

10

1

5

10

1

5

0.0 0.5 1.0 5.0 25.0

232.1 229.0 253.5 268.6 262.5

217.6 212.2 213.9 .= 221.8"* 222.8**

218.9 204.9"" 239.8 282.5" 274.0"

1038.7 1012.7 1169.9 1194.2" 1074.3

622.3 *=

660.4" • 692.0= " 600.3 . ° 6 3 9 . 0 *°

725.1"* 726.9"* 688.7* " 688.1 °* 676.5 *=

609.4 628.9 678.6 701.5" 638.7

494.1"* 507.7"" 594.7" 616.5" 619.8"

590.0 696.7 702.2" 716.7" 722.4 °*.

224.5 195.2 197.8" 183.9" 182.9"

221.7 183.1" 192.2' 140.6 " = .

50.0

270.8

222.1""

243.1"

1037.4

628.6 =.

666.7

616.9

643.5"

716.4 "°.

157.6"

142.0 ==" 125.5 °..

670.8

487.6 °*

157.9

169.6

Males

Females 0.0

187.9

T M

533.8

623.0

226.0 149.7**" 140.2 " j * 102.9*** 98.9*'* 97.7*'*

190.8

221.6*"

606.9="

604.5

0.5

207.8"

245.2 *°

189.0 *=

689.4

564.9 ° * °

647.5

620.8

554.4 "=

601.1

146.3

160.5

137.6"

1.0

200.2

249.4" " *

225.6 " ' *

723.1"

707.8*

731.7'

660.8*

626.1"*

141.7

164.0

127.4"

5.0

216.0"

254.8***

255.5 ° ' *

735.2"

699.5"

757.5*

648.6 . ° .

629.6" "

143.5"

160.6"*

133.4"

25.0 50.0

212.7" 244.8"

247.0" ** 263.3 °

266.0"'" 269.4"

655.1 739.4" 721.9 734.1

162.0

718.9" 726.4"

762.0" 868.2" *"

774.9" 818.1"

694.4" 879.2* "*

785.1" 754.7*

142.4" 132.0"

160.6" " 151.3""

122.1"*' 113.8''*

*Significantly different from c o n t r o l group values (P - 0 . 0 5 ) . * * S i g n i f i c a n t l y different from the 1-dose group values (P = 0 . 0 5 ) . * * * S i g n i f i c a n t l y different from the control group values and the 1-dose group values

(P = 0.05). of liver enzymes were (750 mg/kg and above). levels [ 1 6 , 3 4 , 3 5 , 3 7 , 6 3 ] . sequence of ingestion of result from its industrial

made using anaesthetic doses or high oral doses Significant effects were observed at these high doseThese studies are not directly relevant to the consmall and very small amounts of chloroform as may and laboratory use, as a consequence of atmospheric

T A B L E VII AVERAGE ACTIVITIES OF LIVER SOLUBLE-PHASE ENZYMES OF RATS T R E A T E D WITH C H L O R O F O R M IN G R O U N D N U T OIL Chloroform dose level (mg/kg)

Lactate dehydrogenase

Isocitrate dehydrogenase

Gluco6e-6-phosphate

Number of doses (U/kg body wt)

Number of do6es (U/kg body wt)

Number of doees (U/kg body wt) 1

dehydrogenue

5

10

1

5

10

1

5

10

Ma~s 0.0

6240

6408

6090

284.6

306.0

302.7

77.9

77.7

0.5

6153

6906**

7256***

226.7*

254.4***

316.4"*

72.3

75.0

1.0 5.0 25.0 50.0

5821 6181 7052* 7058*

6804** 7030*** 7272* 7134"

7103"** 7430*** 7724*** 7815"**

261.4 282.3 319.6 412.7"

260.7* 302.6 457.9*** 464.5

423.7*** 419.5"** 460.1"** 444.6*

109.1" 110.2" 104.7" 110.4"

71.3"* 94.4* 98.4* 105.6"

107.2" 92.4** 93.7 95.2

Fem~e$ 0.0 05 10 5.0 25.0 50.0

4091 4086 4702* 5011" 5079* 5092*

5308** 5741"** 5738*** 5772*** 5797*** 6059"**

4328 4769** 5347*** 5392* 5718"** 5794***

384.3 3822 366.3 377.2 506.1" 501.2"

399.0 362.3* 379.8 424.0 485.6* 567.9*

410.9 354.7* 481.3"** 487.7*** 673.7*** 698.5***

328.5 317.7 273.2* 280.4* 254.8* 239.1"

375.2** 360.3** 359.8** 301.0" 311.3"** 358.4**

364.5** 328.9* 276.0* 286.0* 305.9**' 249.3*

93.5** 110.1 * * ~

* Significantly different from c o n t r o l group values (P = 0 . 0 5 ) . ** Significantly different from the 1-dose group values (P = 0 . 0 5 ) . * * * Significantly different from the c o n t r o l group values and the 1-dose group values (P = 0 . 0 5 ) .

32

Aryle~,~ r ~ e

NADPH-Cytochrorne reductase

Cytochrome/>-450

Number of doses (kU/kg body wt)

Number of d o r a (U/kg body wt)

Number of doses (~moles/kg body wt)

1

5

10

1

5

10

1

5

10

5997 7547" 7607"

5995 5940"* 6143""

5910 5776 s " 5074""

462.5 449.9

330.4"* 333.2""

301.8"" 319.6""

128.4 119.2

133.5 139.5

135.1 128.4

439.4

330.0""

332.3*"

120.1

133.5

130.0

7366"

6231"" 6586'"

434.1

322.7"*

345.1''"

118.8

103.3"

140.6""

7406"

5501"" 5990"" 6505

440.5 475.0

319.4"* 358.5 = ' "

347.3''" 344.5"'"

119.1 104.0"

106.2" 105.0"

134.0 136.6"*

5170 5263 5402 5523 5957"'" 6664"'"

4907 5389" 5450" 5744" 6214"*" 7367"'*

261.9 260.0 269.2 327.0" 313.1" 333.5"

270.3 271.9 296.2 340.1" 348.4* 339.2"

293.3"" 280.9 279.1 353.1" 374.9"'" 445.4"'"

132.6 137.4 124.5 103.5" 94.2" 95.9"

153.2"" 144.1 137.1 150.3"" 156.4"" 137.4""

152.4"* 146.9 140.4"" 141.2"" 133.3"' III.0"

7494" 6999" 4624 5186 5225" 5667" 5330" 5712"

pollut-io--n, or from the use of cough mixtures, toothpastes and other proprietary products containing chloroform. For this reason the effects on liver enzymes at very low levels of chloroform intake have been investigated here, i.e. at lower levels than reported before. Also examined was the effect of single as opposed to consecutive daily dosage representing accidental ingestion and repeated exposure to chloroform respectively. Body weights and liver weights showed little consistent change except for slight increases in response to repeated administration with chloroform. These increases seem to be related to the number of doses received as well as to the dose levels. The same applies to the relative liver weights which showed even less effect indicating that liver enlargement in relation to body size is minimal at these dose levels of chloroform. Alanine aminotransferase

Tyrosine aminotransferaBe

Number of dc6es (U/kg body wt)

Number of doses (U/kg body wt)

1

5

I0

1

5

I0

164.1 153.3 161.4 163.3 159.3 160.2

150.3 149.1 153.3 163.1 155.8 153.7

179.4 160.8 155.0" 161.8 155.8" 150.0"

69.2 70.6 89.1" 87.8* 96.9* 89.5*

67.1 75.4* 86.5* 92.0* I05.0" 91.1*

65.9 80.2* 87.4* 91.4" 111.4" I04.6"

174.7 173.5 188.0 198.5" 198.3" 188.6

164.0 168.9 171.4"* 186.5" 166.7"* 167.8

177.2 173.3 181.6 171.1"* 176.8"* 173.9

53.6 70.9* 80.0* 83.3* 77.1' 66.7*

58.7 55.2** 63.8** 68.3*** 69.7* 96.9***

57.3 60.1"* 76.8* 80.6* 80.9* 88.5***

33

In another study in this laboratory, toxic, reparative and adaptive parenchymal changes have been identified histologically in the livers of rats orally intubated with multiple doses of 200 and 300 mg chloroform/kg b o d y wt; similar b u t less prominent effects were present in a proportion of rats dosed with 100 mg/kg b o d y wt. No histological lesions were observed in the livers of animals given dose levels of 0.5--50 mg chloroform/kg b o d y w t although changes in hepatic enzyme activity were found. 3 mitochondrial enzymes involved in oxidative phosphorylation (i.e. glutamate dehydrogenase, succinate dehydrogenase and adenosine triphosphatase) were generally stimulated by chloroform. However, repeated administration reduced the degree of the increase for glutamate dehydrogenase and succinate dehydrogenase, especially at very low dose levels of chloroform, b u t less so as the intake increased. This is due to the combination of 2 effects: {a) direct effect of chloroform on intraceUular metabolism; and (b) an effect probably caused by an adaptation to tissue anoxia which according to Calvert and Brody [64], Moore and Brody [65] and Little and Wetstone [ 7 ], is a secondary effect o f chloroform caused by stimulation of the automatic nervous system leading to hepatic ischaemia. Contradictory evidence as reported by Platt and CockriU [37] and Hecker et al. [16] is probably due to the high doses employed by these authors. The activities of the 2 enzymes which take part in mitochondrial fat catabolism (3-hydroxyacyl-CoA dehydrogenase and acetyl-CoA acetyltransferase) were increased in female rats while males showed only an insignificant inhibition of 3-hydroxyacyl-CoA dehydrogenase after repeated administration. But even in the female animals the initial stimulation of this enzyme diminished after repeated administration. The reason for the behaviour of these 2 enzymes remains unclear. Pyruvate kinase which catalyses the dephosphorylation of phosphoenolpyruvate, an essential step in the glycolytic breakdown of carbohydrates, increased in activity only in females treated with chloroform. This phenomenon may be associated with the incre~ased fatty acid oxidation in this sex because the formation of acyl-CoA from a fatty acid requires energy in the form of ATP. The glycolytic pathway, of which pyruvate kinase is an indicator of activity, is an important source of ATP, and the increased activity in oxidative phosphorylation, another supplier of ATP, is less evident than the increase in fatty acid oxidation activity. The stimulation of amine oxidase activity may be caused by an increased secretion of epinephrine as proposed by Calvert and Brody [64]. Amine oxidase plays an important role in the breakdown of catecholamines. Of all the lysosomal enzymes measured, only ~-glucuronidase did not increase in activity in response to chloroform administration (it was slightly inhibited in the females). The other 3 enzymes of this group which were studied were all stimulated by chloroform and this effect was always stronger in females than in males. Repeated administration had little or no influence on these changes. Stimulation of lysosomal enzymes has been observed in

34

cases of imminent or present tissue damage, i.e. in single cell necrosis as observed in pregnant and lactating rats [ 6 6 ] . It seems quite likely that the changes observed in this experiment foreshadow the massive necrosis observed after exposure to large doses o f chloroform [6,10,18,22,67,68]. The reason for the strange behaviour of j3-glucuronidase cannot be explained. Urate oxidase catalyses the oxidation of uric acid to allantoin and sometimes to other products as well. This is the final step in nucleic acid catabolism in most animals; man is deficient in urate oxidase and the endp r o d u c t of his purine metabolism is uric acid itself. The strong stimulation of this enzyme in the rat after chloroform administration points to an increased breakdown of nucleic acids. Enzymes of the microsomal mixed function oxidase system which were measured were aryl monooxygenase, O-demethylase, N~lemethylase, and the 2 related enzymes, azobenzene reductase and NADPH-cytochrome reductase. All of these with the exception of (aminopyrine) N~iemethylase showed slight stimulation after chloroform intake, an effect which did not change appreciably after repeated administration. N~iemethylase, however, was significantly inhibited b y chloroform and this change was aggravated b y repeated administration. Cytochrome P-450 contents were also reduced after chloroform intake, b u t this effect diminished slightly with repeated administration. According to Gillette [69] these effects are caused by simultaneous proliferation of smooth-surfaced endoplasmic reticulum and destruction of c y t o c h r o m e P-450. Since c y t o c h r o m e P-450 is the co-factor required b y the mixed function oxidases, these enzymes, with the exception of (aminopyrine) N-demethylase, must have been stimulated to a greater extent than their measured activities showed, and this stimulation must have been counteracted b y the partial destruction of e y t o c h r o m e P-450. N-Demethylase often behaves differently from the other mixed function oxidases and therefore our result for this enzyme is n o t surprising. Arylesterase is the name given to a group of microsomal enzymes with low substrate specificities which are involved in the metabolism of a wide variety of drugs [70]. Chloroform ingestion increased the activity o f these enzymes b u t in the males the stimulation disappeared again after repeated administration. The reason for these changes is unknown. Lactate dehydrogenase activity was measured as one example of the large number of enzymes present in the soluble phase of the cell. This enzyme reduces excess pyruvate in skeletal muscle to lactate which is returned t o the liver by the circulating blood. In the liver cell, lactate dehydrogenase re-converts lactate via pyruvate and the Meyerhof-Embden pathway to glucose (Cori or lactic acid cycle). Changes in the activity of this enzyme give an indication of changes in the pyruvate c o n t e n t of the tissues which in turn reflect changes in all the major catabolic pathways [71]. Lactate dehydrogenase activity increased after chloroform intake and a further increase resulted from repeated administration. This stimulation is probably due to increased breakdown of carbohydrates and protein in muscle which

35

both contribute to the pyruvate pool of the cell. Stimulation of the marker enzymes selected for these 2 pathways as well as the citric acid cycle was also found and a further point in favour of this explanation is that these enzymes show a similar response to repeated administration and similar sex differences. However, administration of chloroform in larger doses leads to inhibition of lactate dehydrogenase according to Platt and Cockrill [37]. Isocitrate dehydrogenase is one of the enzymes of the citric acid cycle which links the major metabolic pathways. Chloroform ingestion increased its activity indicating a stimulation of this central part of intermediary metabolism. The most important enzyme of the hexose phosphate shunt, glucose-6-phosphate dehydrogenase, showed a particularly strange reaction to chloroform administration. While in the males it was greatly stimulated after the first dose, in the females it was significantly inhibited under these conditions. Repeated administration caused additional inhibition in both sexes and this change was considerably greater in the males. After 10 doses, glucose-6-phosphate dehydrogenase activity was significantly decreased in the females and about normal in the males, indicating t h a t in the former the turnover via the hexose phosphate shunt was reduced, while in the latter it was hardly affected. Prolonged administration would probably have led to inhibition in the males too. This, together with the reduction in cytochrome P-450 contents, seems to be a contributing factor to the inhibition of the microsomal mixed function oxidases observed after the administration of large doses of chloroform since glucose-6-phosphate dehydrogenase is the main supplier of NADPH required for the action of these enzymes. Of the 2 aminotransferases studied, the activity of alanine aminotransferase decreased in the males, but only after 10 doses while in the females an initial stimulation disappeared after 10 doses. This shows that the effect of repeated administration is identical in both sexes while a single dose affects only the females. Tyrosine aminotransferase activity, however, was stimulated in both sexes and in the males this effect increased with repeated administration. Since tyrosine aminotransferase is completely under the control of adrenal cortical hormones [72,73] it appears that chloroform causes increased secretion of adrenal cortical as well as of adrenal medullary hormones. In this study, the effects of chloroform on the activities of rat liver enzymes were generally dose-related but, as expected, the effects did n o t show a linear trend with continuing dosage. It is concluded that the ingestion of chloroform even in very small amounts can lead to distinct changes in cellular metabolism. These changes are sometimes the same as, but sometimes different from, those seen with larger and anaesthetic doses. In most cases a transition between these 2 types of effects can be seen. Chloroform has therefore to be regarded as toxic even in very low concentrations. REFERENCES 1 L. Loeffler, Arch. Pathol. Anat., 269 (1928) 771.

36

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2~2 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

S. Goldschmidt, H.M. Vats and I.S. Radvin, J. Clin. Invest., 18 (1939) 277. H.L. Sheehan, Br. J. Anaesth., 22 (1950) 204. W.M. Jones, G.K. Margolis and C.R. Stephen, Anaesthesiology, 19 (1958) 715. B. Kylin, H. Reichard, I. Sumegi and S. Yllner, Acta Pharmacol. Toxicol., 20 (1963) 16. M. Siess, B. Schmidt, H. Oehmig and E. Kirchner, Bruns Beitr. Klin. Chir., 206 (1963)461. D.M. Little and H.J. Wetstone, Anaesthesiology,25 (1964) 815. S. Sherlock,Proc. R. Soc. Med., 57 (1964) 305. A. Galindo, L.D. MacLean, R.G.B. Gilbert and G.F. Brindle, Can. Anaesth. Soc. J., 12 (1965) 443. C. Morgenstern, J. Haumann, L. Cossel, B. Wohlgemuth and D. Kunze, Arzneim.-Forsch., 15 (1965) 349. W.D. Wylieand H.C. Churchlll-Davidson,A Practice of Anaesthesia, 2nd Edn., LloydLuke, London, 1966. W.A.Wolff,W.V. Lumb and M.K. Ramsay,Am. J. Vet. Rcs., 28 (1967) 1363. G. Feiae,Z. Gesamte. Exp. Med., 147 (1968) 190. M.A.Ramadan and M.I.A. Ramadan, Acta Histochim., 34 (1969) 310. E. Thorpe, C. Gopinath, R.S. Jones and E.J.H. Ford, J. Pathol., 97 (1969) 241. D. Hecker, M. Meyer, K. Lohs and G. Wlldner, Acta Histochem., 41 (1971) 293. J. Howlandand A.N. Richards,J. Exp. Med., 11 (1909) 344. G.H.Whippleand J.A. Sperry, Bull. Johns Hopkins Hosp., 20 (1909) 278. R.W. Brauer, G.F. Leong and R.J. Holloway, Am. J. Physiol., 200 (1961) 548. G. Corssen, R.B. Sweet and M.B. Chenoweth, Anaesthesiology, 27 (1966) 155. G. Corssen, Cytotoxic effects of halogenated anaesthetics,in B.R. Fink (Ed.), Toxicity and Anaesthetics, Proceedings of a Research Symposium May 1967, Seattle, Williams and Wilkins, Baltimore, p. 50. K.L. Scholler, Anaesthetist, 15 (1966) 145. K.L. Scholler, Experientia, 23 (1967)652. J. McG. Imray, B.R. Kennedy and S.J. Kilpatrick, Anaesthesia, 19 (1964) 33. W.N. Rollason, Proc. R. Soc. Med., 57 (1964) 307. M.J. Kirwan, J.W. Dundee and D.W. Neill, Anaesthesia, 20 (1965) 66. C.D. Klaassen and G.L. Plaa, Toxicol. Appl. Pharmacol., 9 (1966) 139. P.J. Gehring, Toxicol. Appl. Pharmacol., 13 (1968) 287. K.L. Scholler, Fortschr. Med., 88 (1970) 781. K.L. Scholler, Br. J. Anaesth., 42 (1970) 603. K.L. Scholler, Anaesthetist, 20 (1971) 149. K.L. Scholler, E. Miiller and U. yon Plehwe, Arzneim.--Forsch., 20 (1970) 289. M. Ojiri, Masui, (Jpn. J. Anaesthesiol.), 14 (1965) 954. J.V. Dingell and M. Heimberg, Biochem. Pharmacol., 17 (1968) 1269. H.A. Sasame, J.A. Castro and J.R. Gillette, Biochem. Pharmacol., 17 (1968) 1759. C.D. Klaassen and G.L. Plaa, Biochem. Pharmacol., 18 (1969) 2019. D.S. Platt and B.L. Cockrill, Biochem. Pharmacol., 18 (1969) 445. W.C. Schneider and G.H. Hogeboom, J. Biol. Chem., 183 (1950) 123. G.H. Hogeboom and W.C. Schneider, J. Biol. Chem., 204 (1953) 233. R.J. Pennington, Biochem. J., 80 (1961) 649. M.E. Pullman, H.S. PenefskT, A. Datta and E. Racker, J. Biol. Chem., 235 (1960) 3322. K. Decker, Dissertation, University of Munich, 1955. J.R. Stern in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology I, Academic Press, London, 1955, p. 581. H.J. Biicher and G. Pfleiderer in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology I, Academic Press, London, 1955. p. 435. H. Weissbach, T.E. Smith, J.W. Daly, B. Witkop and S. Udenfriend, J. Biol. Chem., 235 (1960) 1160.

37

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

38

A.C. Allison and K. Sandelin, J. Exp. Meal., 117 (1963) 879. A.J. Anderson, Biochem. Pharmacol., 17 (1968) 2253. O.A. Bessey, O.H. Lowry and M.J. Brock, J. Biol. Chem., 164 (1946) 321. A.B. Roy, Biochem. J., 53 (1953) 12. E. Praetorius, in H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Verlag Chemie--Academic Press, Heidelberg, 1963, p. 500. T.E. Gram, L.A. Rogers and J.R. Fouts, J. Pharmacol. Exp. Ther., 155 (1967) 479. R. Kato and J.R. Gillette, J. Pharmacol. Exp. Ther., 150 (1965) 279. R.E.. McMahon, H.P. Culp, J. Mills and F.J. Marshall, J. Med. Pharm. Chem., 6 (1963) 343. J. Cochin and J. Axelrod, J. Pharmacol. Exp. Ther., 125 (1959) 105. P.H. Hernandez, J.R. Gillette and P. Mazel, Biochem. Pharrnacol., 16 (1967) 1859. M.M. Nachlas and A.M. Seligman, J. Biol. Chem., 181 (1949) 343. T. Omura and R. Sato, J. Biol. Chem., 239 (1964) 2379. H.U. Bergmeyer, E. Bernt and B. Hess, in H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Veflag Chemie-Academic Press, Heidelberg, 1963, p. 736. G.W.E. Piaut and S.C. Sung, J. Biol. Chem., 207 (1954) 305. A. Komberg and B.L. Horecker in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology I, Academic Press, London. 1955, p. 323. H.U. Bergmeyer and E. Bernt in H.U. Bergrneyer (Ed.), Methods of Enzymatic Analysis, Verlag Chemic-Academic Press, Heidelberg, 1963, p. 846. F. Rosen, H.R. Harding, R.J. Millholland and C.A. Nichol, J. Biol. Chem., 238 (1963) 3725. E. Leuwenkroon-Strosberg, L.H. Laasberg and J. Hedley-Whyte, Biochim. Biophys. Acta, 295 (1973) 178. D.N. Calvert and T.M. Brody, Am. J. Physiol., 198 (1960) 664. K.E. Moore and T.M. Brody, Am. J. Physiol., 198 (1960) 677. R. Wilson, B.H. Doell, W. Groger, J. Hope and J.B.M. Gellatly, The physiology of liver enlargement, in F.J.C. Roe (Ed.), Metabolic Aspects of Food Safety, Blackweli Scientific Publications, Oxford and Edinburgh, 1970, p. 363. W.F. von Oettingen, The halogenated hydrocarbons, in Toxicity and Potential Dangers No. 414 U.S. Department of Health, Education and Welfare, Washington, D.C. 1955. E.S. Reynolds and A.G. Yee, Lab. Invest., 16 (1967) 591. J.R. Gillette, Enzyme induction in laboratory animals and its relevance to food additive investigation, in F.J.C. Roe (Ed.), Metabolic Aspects of Food Safety, Blackwell Scientific Publications, Oxford and Edinburgh, 1970, p. 282. D.V. Parke, The Biochemistry of Foreign Compounds, Pergamon Press, Oxford, 1968. H.A. Harper, Review of Physiological Chemistry, Blackwell Scientific Publications, Oxford and Edinburgh, 1967, p. 238. E.C.C. Lin and W.E. Knox, Biochirn. Biophys. Acta, 26 (1957) 85. E.C.C. Lin and W.E. Knox, J. Biol. Chem., 233 (1958) 1186.