Toxicity of cyclohexanone oxime

Toxicity of cyclohexanone oxime

FUNDAMENTAL AND APPLIED TOXICOLOGY 5, 117- 127 ( 1985) Toxicity of Cyclohexanone Oxime I. Hematotoxicity following Subacute Exposure in Rats’,...

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FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

5, 117-

127 ( 1985)

Toxicity of Cyclohexanone

Oxime

I. Hematotoxicity following Subacute Exposure in Rats’,*

MICHAEL J. DERELANKO,~ SHAYNE C. GAD, WILLIAM J. POWERS, SEBASTIAN MULDER, FRANCES GAVIGAN, AND PETER C. BABICH Department of Toxicology, Allied Corporation, Morristown. New Jersey 07960 Toxicity of Cyclohexanone Oxime. I. Hematotoxicity following Subacute Exposure in Bats. DERELANKO,

M.

J., GAD,

S. C.,

POWERS,

W.

J., MULDER,

S., GAVIGAN,

F., AND

BABICH,

P. C. (1985). Fundam. Appl. Toxicol. 5, 117-127. Cyclohexanone oxime (CHO) was given po to male and female Fischer 344 rats at dose levels of 10, 25, 75, 150, and 300 mg/kg, five times a week for a period of 2 weeks. Control animals received distilled water. All animals given intermediate dose levels (10, 25, 75, and 150 mg/kg) and one half of the animals which were dosed at the high dose (300 mg/kg) as well as one half of the controls were terminated 14 days after administration of the first dose. The remaining rats received no treatment for an additional 14 days and were sacrificed on Day 28 of the study (recovery phase). Dose-related decreases in erythrocyte number, hemoglobin, and hematocrit, with an accompanying increase in mticulocytes and circulating nucleated erythrocytes, were observed in both sexes at Day 14. Methemoglobin levels, determined only at the high dose, were elevated in both sexes at this time. Splenomegaly and hepatomegaly were observed in both sexes at 14 and 28 days. Histopathologicrd examination of the spleen and bone marrow revealed dose-related erythroid hyperplasia at 14 days which subsided by Day 28. The above effectswere more pronounced in males. Erythrocyte numbers were only slightly depressed and reticulocytes mildly elevated in males at Day 28. Hematological values were not statistically different from controls in females at this time. These results suggest that CHO induces oxidative damage to the erythrocyte, resulting in a hemolytic anemia accompanied by increased erythropoiesis. The toxic effects appear reversible upon CeSSatiOn of exposure. 0 1985 society of Toxicology.

Cyclohexanone oxime (CHO) is used as an intermediate in the synthesis of caprolactam which in turn is utilized in the production of nylon 6. CHO is produced from the reaction of hydroxylamine with cyclohexanone. CHO is handled in a captive process which limits the risk of worker exposure. However, the large volumes handled yearly necessitates a thorough understanding of the toxicity of this material. Several reports in the literature have described a decrease in erythrocyte mass and increased methemoglobin levels in rabbits ’ This study was supported in part by the Industrial Health Foundation. 2 Presented, in part, at the 66th Annual Meeting of the Federation of American Societies for Experimental Biology, New Orleans, La., April 15-23, 1982. 3 To whom requests for reprints should be addressed.

and rats following acute oral (Lomonova, 1966) and subchronic inhalation (Tsulaya et al., 1975) exposure respectively, to CHO. However, histopathological examination and clinical analyses were limited or lacking in these studies. Tsulaya et al. (1975) reported the presence of anemia and reticulocytosis in workers exposed to CHO. Data relative to these findings were not presented. The purpose of the present investigation was to confirm and to expand the above findings on the hematotoxic effects of CHO and to determine if the reported hematotoxicity of CHO is reversible upon cessation of exposure. MATERIALS

AND

METHODS

Material. A sample of CHO was supplied by tbe Hopewell Chemical Plant of the Allied Chemical Company, a Division of the Allied Corporation. Dosing 117

0272-0590185 $3.00 Copyright 0 I985 by the Society of Toxicolcgy. All rigbls of reproduction in any form reserved.

118

DERELANKO

solutions were prepared fresh weekly as follows: A stock I .5% solution of CHO (w/v) was prepared by heating an appropriate amount of CHO in distilled water with constant stirring. The temperature of the solution was not allowed to exceed 60°C. Dosing solutions of lesser concentrations were prepared by dilution of the 1.5% stock solution. A high-performance liquid chromatography stability analysis, performed prior to the start of the study, indicated that CHO was stable for at least 1 week in aqueous solution at concentrations of 0. I to 1.5%. Animals. Fifty male and fifty female Fischer 344 rats received from Charles River Breeding Laboratories, Kingston, New York, were used in this study. AII animals were identified by numbered ear tags upon arrival and quarantined for 2 weeks prior to dosing. General health of the rats was assessed by outward appearance and weight gain during this time. The animals were housed individually in suspended cages with wire-mesh bottoms in a room maintained at a temperature of 72“ + 2”F, a humidity of 50 + 5%, and a light/dark photoperiod of 12 hr. Animals were allowed water from an automatic system and Purina Rodent Chow 5001 ad libitum except during dosing and other procedures requiring handling. Experimental design. Animals were randomly assigned by sex to groups of five animals each by means of a computer program which utilizes analysis of variance (ANOVA) to ensure that the mean body weight of each group within a sex do not statistically differ at the beginning of the study. At the initiation of the study, male rats weighed 212 to 284 g while females weighed 146 to 178 g. All animals were weighed immediately prior to each dosing and the dose volume was based on that weight. Dosing was by gavage using a 16-gauge feeding needle. CHO was administered five times a week for 2 weeks at dose levels of 10, 25, 75, 150, and 300 mg/kg and at a dose volume of 20 ml/kg for tbe high dose level and IO ml/kg for the remaining dose levels. Control rats received distilled water at a volume of 20 ml/kg. All animals were closely observed for signs of toxicity twice each day until completion of the study. Body weights were taken daily throughout the study. All animals given intermediate dose levels of CHO (10, 25, 75, and 150 me/kg) and one half of the animals which were dosed at the high dose level (300 mg/kg), as well as one half of the controls, were sacrificed 14 days after administration of the first dose. The remaining test and control rats received no treatment for an additional 14 days and were sacrificed on Day 28 of the study. These rats, designated for the recovery phase of the study, were weighed daily for the t&t 14 days after initial dosing and approximately every other day until Day 28. Changes in body weight during the study were calculated from these measurements using the Day 0 weight as baseline. Neurotoxicological assessment was accomplished by use of a neurobehavioral screen procedure (Gad, 1982) on the day of initial dosing and subsequently on Days 1, 7, 14, and 28.

ET AL. Clinical determinations. Blood and urine samples were collected from each animal on the morning of the day of sacrifice. Animals were placed in stainless-steel metabolism cages and urine was collected over a 3-hr period into amber-colored bottles placed in ice. The animals were deprived of food and water during the collection period. Urine parameters were measured within I hr following the end of the collection period. The urine was assayed for the presence of urobilinogen4 and hemoglobin? Blood was collected under ether anesthesia by intraorbital or aortic puncture depending on the parameter measured. Hematological parameters determined on EDTA-anticoagulated blood obtained from all groups via intraorbital puncture included erythrocyte count’; leukocyte count5; total hemoglobin5; hematocrit5; mean corpuscular volume, hemoglobin, and hemoglobin concentration’; and platelet count.6 Leukocyte differential counts were determined manually from Wright-Giemsa stained peripheral blood smears. Free serum hemoglobin’ was assayed using the o-tolidine reaction (Bauer et al., 1968). Methemoglobins levels were determined using an IL-282 Co-oximeter’. Serum total bilirubin’ values were assayed with a Rotochem Parallel Fast Analyzer” using Worthington” reagents. Glucose-6-phosphate dehydrogenase activity’ was measured using an Erythrozyme12 test kit. Erythrocyte catalase activity’ was assayed using a modification of the sodium perborate method of Feinstein as described by Tarlov and Kellermeyer ( I96 1). Reticulocyte counts were determined manually from new methylene blue stained peripheral blood’ smears. A method originally described by Fruhman and Gordon (1955) and modified by LoBue et al. (1963) was utilized to determine the femoral bone marrow myeloid/erythroid ratio. Briefly, this method consists of preparing a femoral bone marrow suspension in a hypotonic solution of fetal calf serum and distilled water (2:l). Smears were made from this suspension, stained with o-tolidine and counterstained with Wright-Giemsa. A total of 500 cells were differentiated by microscopy. The marrow from the right femur was used exclusively. Pathology. Necropsy was performed on each animal 14 days after initiation of dosing or on Day 28 in the case of the recovery groups. Animals were terminated

4 Hemastix and Urobilistix, Ames Company, Elkhart, Ind. ’ Hemac, Ortho Instruments, Westwood, Mass. 6 Ultra-Flow 100, Clay-Adams, Parsippany, N.J. ’ Blood was collected via an intraocular technique. 8 Blood was collected via aortic puncture. 9 Instrumentation Laboratory, Inc., Lexington, Mass. lo Rotochem Parallel Fast Analyzer, American Instrument Company, Chicago, III. ” Worthington Biochemical Corporation, Freehold, NJ. I2 Biomedix, Princeton, N.J.

HEMATOTOXICITY

OF CYCLOHEXANONE

by exsanguination under Ketalar” (ip) anesthesia. Lungs, liver, spleen, kidneys, testes, sternum with marrow, and portions of the gastrointestinal tract were removed, fixed in 10% Formalin, and stained with hematoxylin-eosin for histopathological examination. Organs were weighed at sacrifice and organ weight/body ratios were calculated from these measurements and terminal body weights. Statistical analyses. Statistical analyses was based on a decision-tree scheme for selecting statistical procedures as described by Gad and Weil (1982). The results of quantitative continuous variables, such as body weights, organ weights, and hematological and clinical parameters, with the exceptions stated below, were intercompared for test versus control groups by employing the following &&t&l tests:Bartlett’s homogeneity of variance, analysis of variance, and Duncan’s multiple range test. The latter was used to delineate which groups differed from controls where the F for analysis of variance was significantly large. If Bartlett’s test indicated heterogeneous variance or where data were ranked or suspected to be nonparametric, the Kruskall-Wallis nonparametric analysis of variance and the Wilcoxan rank sum tests were employed. Parameters requiring nonparametric analysis included reticulocyte, absolute monocyte, and absolute eosinophil counts; and anisocytosis, poikilocytosis, polychromasia, and Howell-Jolly body scores.

RESULTS Mortality

and General Signs of Toxicity

No deaths occurred in the male rats regardless of the dose level of CHO administered. One of the female rats which received CHO at a dose level of 300 mg/kg died on Day 9 of the study. No significant gross signs of toxicity were noted of this animal during the 8 days preceding death. The death of this animal, most likely unrelated to CHO-induced toxicity, possibly resulted from physical injury which was induced on Day 8 during the dosing procedure. No mortality occurred in the remaining CHO-treated female rats during the duration of the study. No significant gross signs of toxicity were observed in either sex during the study which could be correlated with CHO administration. The only neurotoxic effect which could be associated with CHO administration was an increase in lacrimation observed sporadically I3 Parke-Davis, Morris Plains, N.J.

OXIME

119

in routine clinical observations performed during the dosing phase of the study. This effect was more intense in females than in males. However, the increased lacrimation did not occur in either sex in a dose-related manner. Moreover, while none of the males which received distilled water exhibited increased lacrimation, 70% of the control females displayed this effect. This suggests that the increase in lacrimation noted in both sexes was most likely not directly related to a toxic property of CHO but probably resuhed from stress induced by the dosing procedure. No signs of neurotoxicity were observed during the recovery phase of the study. No significant differences in mean body weights were noted between CHO-treated animals and controls during the dosing and recovery phases of the study. Hematological

and Clinical Parameters

HematologicaI values of male and female CHO-treated and control rats 14 days after initiation of dosing are presented in Tables 1 and 2, respectively. A dose-related decrease in erythrocyte number, hematocrit, and total hemoglobin occurred in both sexes. The decrease was statistically significant in the males (p < 0.01) at CHO dose levels of 25 mg/kg and higher. In the females, only the erythrocyte count was statistically reduced at the 300-mg/kg dose level. The decrease in erythrocyte-related parameters was accompanied in both sexes by a dose-related increase in mean corpuscular volume, hemoglobin, and hemoglobin concentration. A dose-related increase in both reticulocytes and circulating nucleated erythrocytes occurred in both sexes. The degree of polychromasia, anisocytosis, and poikilocytosis was greater with increasing dose levels of CHO. Howell-Jolly bodies were present in erythrocytes of both male and female CHO-treated rats. Heinz bodies were observed in etythrocytes of both sexes at dose levels of 150 and 300 m&kg. Total leukocyte counts (corrected for circulating erythroblasts) were significantly ele-

2

Hematocrit Total hemoglobin Mean corpuscular

IV/mm3 103/mm3

0.01 + 0.022 4.6 f 0.46 649 f 112.3

0.1 f 0.11 6.9 + 0.62’ 704 + 47.4

0.0 0.7 f 0.42

0.0 0.2 + 0.12

%

SCOd

scored

Scored

25

Treatment

f o.39c 0.6 1.4 2.4

0.4 f 0.19’ 7.2 2 l.lOb 802 + 108.6

0.0 1.8 f 1.88’

31.50

18.0 + 0.31

57 f 0.5

MALE

75

weeks)

WBC,

present.

0.6 k 0.33b 7.6 + l.lOb 853 + 49.3

1.2 3.6 k 0.70b

32.3 + 0.67’ 0.8 1.4 2.4

19.2 -t 0.59b

59 f 1.2b

DOSING

4 = >8 cells/100

WBC.

1.2 + 0.32’ 7.3 + 0.81 b 757 + 155.3

1.8’ 5.4 f 1.84b

32.9 + 0.68b 2.0’ 2.2c 3.4

20.0 + 0.49b

60 f 0.9’

7.07 5 0.322” 43.0 + 2.08’ 14.1 + 0.60b

150

RATS 14 DAYS AFTER INITIAL

7.51 + 0.323” 44.6 + 1.43b 14.4 + 0.63b

(SX/week/2

AND CONTROL

8.12 + 0.237b 46.5 + 1.03’ 14.6 k 0.30’

1

b Significantly different from control, p < 0.0 1 ’ Signiticantly different from control, p < 0.05. dO = parameter not present; 1 = minimally present; 2 = slightly present; 3 = moderately present; 4 = markedly ’ = Not applicable. ‘0 = no cells containing parameter present; 1 = 1-2 cells/100 WBC, 2 = 3-5 cells/100 WBC; 3 = 6-8 cells/100

Leukocvtes

31.4 f 0.52 0.4 1.0 1.6

31.3 k 0.37 0.08 0.6 1.0

%

SCOR?d

17.6 + 0.41

17.6 + 0.34

56 f 0.5

56 + 0.4

10

(CHO)-TREATED

9.01 + 0.162 50.4 + 0.55 15.9 + 0.15

water PO)

OXIME

9.06 k 0.265” 51.0 + 1.57 16.0 + 0.57

(20 ml/k&

Distilled

VALUES OF CYCLOHEXANONE

pg/RBC

pm’

[email protected]/mm”

ErythroCyt=

Mean corpuscular hemoglobin Mean corpuscular hemoglobin concn Poikilocytosis AIliSOCytOSiS Polychromasia Howell-Jolly bodies Reticulocytes Circulating nucleated

Units

Parameter

HEMATOL~CXAL

TABLE

1.3 f 0.33b 6.6 + 1.506 779 f 244.8

2.0’ 8.3 + 2.47’

33.8 + 0.129b 1.4= 2.8’ 3.8’

21.2 zk 0.63’

62 + 0.8b

6.16 + 0.216b 38.7 f 1.69’ 13.1 + 0.596

300

F

3

3o

P

u g

‘J 0

% 106/mm3 Is

Etythrocytes Hematocrit Total hemoglobin Mean corpuscular volume Mean corpuscular

score’ %

Id/mm3 103/mm3 103/mm3

Circulating nucleated erythrocytes Leukocytes Platelets

0.05 AZ 0.03 5.10 + 1.53 502 f 62.2

0.4 0.7 + 0.32

“Mean+ 1 SD. b Significantly different from control, p -z 0.01, c Siiificamly different from control, p < 0.05. dO = parameter not present; 1 = minimally present; e Not applicable. 10 = no cells containing parameter present; 1 = l-2

SCOEd

31.2 f 0.70 0.2’ 0.8 1.2

56 Scored scored

Howell-Jolly bodies Reticulocytes

18.6 + 0.62

59 f 1.1

7.26 + 0.964” 43.40 6.13 13.6 f 2.03

Pidmc

hemoglobin Mean corpuscular hemoglobin concn Poikilocytosis Anisoqtosis Polychromasis

pm3

Units

Parameter

celIs/lOO

10

(CHO)-TREATED

WBC,

2

25

Treatment

2 0.68’ 0.8 1.8 2.4”

3 = moderately

WBC;

15

@g/kg,

weeks)

4 = markedly

WBC;

present.

0.9 * 0.19c 8.0 + 0.79’ 799 f 147.9”

1.6 4.0 + l.95b

31.5 k 0.45’ 0.4 2.4” 2.6’

20.2 + 0.4’

63 + 0.5’

+ 0.27’

WBC.

1.4 f 0.55’ 7.5 f 0.5Ob 832 + 103.5

2.0’ 5.8 f 1.45”

31.9 + 0.64’ l.4c 2.4c 3.2’

20.60

64 f 0.7’

42.8 6.64 f 0.193 1.25” 13.7 * 0.51

150

INITIAL

4 = 18 cells/100

PO)

RATS 14 DAYS AFIZR

6.67 42.8 +f 0.373 2.13’ 13.7 + 0.52

CHO

3 = 6-8 cells/100

present;

0.6 ct_ 0.29’ 7.5 k 0.8gb 828 k 54.2”

1.2 3.9 +- 1.57b

31.30

19.3 + 0.62’

61 + l.lb

FEMALE

(W/week/2

AND CONTROL

7.05 43.5 2z!c 0.294 1.486 13.6 + 0.37

2 = 3-5 cells/100

present;

0.06 + 0.06 6.7 AZ 0.43’ 725 + 33.8

0.0 0.4 + 0.22

30.9 + 0.57 0.4 0.6 1.2

18.5 f 0.34

59 + 0.5

8.34 49.9 ++ 0.397b 2.63b 15.4 f 0.75

2 = slightly

Distikd water (20 mk/kg, PO)

HEMATOL~CICAL VALUES OF CYCLOHEXANONE OXIME

TABLE DOSING

1.8 f 0.25c 5.4 + 0.50” 824 f 91.6

2.3c 7.0 + 0.50’

32.3 _t 0.65 c 0.8 2.5d 4.0’

21.1 + o.40b

65 zk 0.6b

6.08 39.6 f+ 0.143* 1.02 12.8 ? 0.29

300

5 Fl

9

3 5 I3

a

z 3 3

8

il%

122

DERELANKO

ET AL.

hemoglobin and urobilinogen levels were within the normal range for both male and female CHO-treated animals.

vated in CHO-treated rats of both sexes but the increase was not dose related. The elevated total leukocyte counts were entirely associated with increased numbers of circulating lymphocytes. Platelet numbers were elevated in CHO-treated animals for both sexes. However, the increased numbers were not dose related and were only statistically different from controls in the females. Of those parameters measured only at the 300-mg/kg dose level (Table 3), blood methemoglobin levels were significantly elevated 0, < 0.01) in both CHO-treated males and females 14 days after initial dosing. Glucose6-phosphate dehydrogenase levels were significantly greater in the females (J < 0.01) while being slightly (although not significantly) elevated in the males. Erythrocyte catalase activity was slightly elevated in both sexes but statistically greater (p < 0.01) than the control values only in the males. Serum total bilirubin levels were statistically elevated in the males only. Free serum hemoglobin levels were not greater than control values in either sex and were statistically lower (p < 0.01) in the CHO-treated male rats. Urine

Pulhological

Findings

Splenomegaly was a common finding at necropsy in CHO-treated rats of both sexes. Cervical lymph nodes were abnormally dark in color in those animals which received CHO at dose levels of 150 and 300 mg/kg. No other gross pathological signs indicative of compound-related toxicity were apparent at necropsy. Absolute and relative spleen weights were significantly (p < 0.01) greater than control values in both males and females at CHOdose levels of 25 mg/kg and greater (Table 4). An increase in relative liver weight occurred in CHO-treated male rats at all dose levels and was statistically greater than control values at dose levels of 75 mg/kg and above (Table 4). A significant increase in liver weight (absolute and relative) occurred in females only at the 300-mg/kg dose level. Histopathological examination revealed

TABLE

3

CLINICAL VALUES OF CYCLOHEXANONE OXIME (CHO)-TREATED AND CONTROL RATS 14 DAYS AFTER INITIAL DOSING Treatment (W/week/Z weeks) Males Distilled water Units

Parameter

Metbemoglobin Femoral marrow myeloid/ e&mid ratio GhWJSA-PllOSplUUe deha (rrythmcyte) ~~kd=Yw Total serum bilbubin Free scmm hemoglobin

w Unit&Hb meq NaBox decomposed

(5 min) mgsb m%R,

‘MeanfISD. b Signiiicantly differentfrom control, p < 0.01. cSignificantIyd&-em from control, p c 0.05.

Females

(20mB8, PO)

CHO (300 m&8, po)

1.5 k 0.38" 1.85 2 0.356

4.4 + 1.056 0.41 + 0.0176

22.03 " 4.208

23.60 k 2.828

Distilled water PO)

(20wk

CHO (3Wmgnc&~o)

1.2 * 0.30 1.65 f 0.396

2.9 + 0.176 0.38 f 0.099b

16.83 f 2.221

22.73 k 0.72Sb

0.21 2 0.008

0.24 f 0.021c

0.23 2 0.022

0.25 f 0.013

0.1 2 0.0

0.22 * 0.0476 6.00 + 1.740"

0.08 k 0.045 8.1 f 3.40

0.10 * 0.0 7.8 + 1.97

10.3 * 1.66

8 mp/g g mtif3

Spleen weight Spleen ratio Liver weight Liver ratio

0.42 2.55 5.51 33.76

0.50 1.86 8.81 32.70

+ k + -t

0.039 0.153 0.313 1.067

_t 0.065’ + 0.127 + 1.120 +- 1.350

0.41 2.34 5.43 31.24

0.56 1.95 9.91 34.23

OF CYCLOHEXANONE

Distilled water (20ml/kg, PO)

WEIGHTS

“Mean&lSD. b Significantly different from control, p < 0.01. c Organ weight/body weight ratio (relative weight). d SignificantIy different from control, p c 0.05.

B wJg B mtig

Spleen weight Spleen ratio’ Liver weight Liver ratio

Units

SPLEEN AND LIVER

0.054 0.151 0.506 1.826

f 0.030 zk 0.141 zk 0.233 k 1.165

f k + f

10

OXIME

AND CONTROL

0.5 I 3.14 5.27 32.64

0.67 2.54 9.05 34.14

* 0.034h + 0.278* 3~ 0.172 + 1.933

2 0.024” + 0.127h + 0.755 +_ 0.929

25

0.91 3.34 10.06 36.96

0.63 3.65 5.50 32.06

Females

Males

weeks)

ANIMALS

PO)

r 0.043h + 0.120n + 0.287 & 1.929

0.62 3.66 5.59 32.96

1.02 3.82 9.74 36.34

DOSING

+ f * +

0.050* 0.306h 0.301 1.659

f 0.085 h f 0.251 h + 0.865 -t_ 1.867’+

150

14 DAYS AFI’ER INITIAL

k 0.070h + 0.184” -+ 0.632 -t 1.186h

CHO Ow/k 75

Treatment (5x/week/2

(CHO)-TREATED

TABLE 4

0.76 4.51 6.02 35.51

1.09 4.34 9.67 38.36

0.093h 0.284’ 0.752 0.908”

A 0.043h -e 0.251” + 0.556d AI 1.120d

f f + +

300

9 0 n 2 F! 8 53 4 8 m x0

8

2 E 5 3

124

DERELANKO

compound-related effects in both spleen and bone marrow. The spleen of CHO-treated males and females was congested with foci of nucleated erythroblasts and macrophages containing increased hemosiderin. Hemosiderin was confirmed using Gomori’s modification of Perl’s Prussian blue reaction. The sternal marrow of both sexes displayed erythroblastic hyperplasia with an accompanying decrease in fatty marrow. The femoral marrow myeloid/erythroid ratio was significantly 0, < 0.01) reduced in both CHO-treated males and females (Table 3) at a dose level of 300 mg/kg (parameter not measured at lower dose levels). Recovery Phase One half of the rats of both sexes from the high dose and control groups were not sao rificed on Day 14 of the study, but were observed for an additional 2-week period during which time these animals received no additional exposure to CHO. No gross signs indicative of toxicity were noted in these animals during the 2-week recovery phase. No difference in body weight parameters occurred between CHO-treated and control animals during this time. Hematological values measured on Day 28 for CHO-treated and control rats of both sexes are presented in Table 5. Erythrocyte counts were slightly decreased @ < 0.01) in CHO-treated males compared to controls. Other erythrocytic indices, such as total hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, were mildly elevated (p < 0.01) compared to controls at this time. Male reticulocyte counts were elevated on Day 28. However, this represented only a 2-fold increase over control values compared to a T-fold increase noted on Day 14. Other related parameters such as circulating nucleated erythrocyte numbers, as well as the degree of anisocytosis, poikilocytosis, and polychomasia, were at near normal levels in the males on Day 28. Howell-Jolly bodies

ET

AL.

were not observed in the circulating erythrocytes at this time. Leukocyte and platelet numbers were normal. In the CHO-treated female rats evaluated on Day 28, erythrocyte count, hematocrit, total hemoglobin, and reticulocyte numbers were not statistically different from control values. A slight, but statistical increase in mean corpuscular volume, hemoglobin, and hemoglobin concentration was noted at this time. All other hematological parameters were similar to control values. Methemoglobin levels of both CHO-treated males and females were not significantly different from control values on Day 28 of the study (Table 6). As also presented in Table 6, femoral marrow erythropoiesis was reduced as evidenced from the increased myeloid/ erythroid ratios for both sexes. Glucosedphosphate dehydrogenase activity was still elevated in both male and female CHOtreated animals at this time. Cataiase, total serum bilirubin, and free serum hemoglobin determinations were not performed on the recovery animals. Both spleen and liver weights of CHOtreated males were significantly greater than those of controls on Day 28 of the study (Table 7). Liver weights of CHO-treated females were statistically greater than control weights. However, these elevated organ weights represented only a slight increase over the norm at this time. DISCUSSION It is well known that exposure to certain chemicals and drugs can damage erythmqtes, leading to their selective destruction by the reticuloendothelial system (Beutler, 1969; Weed and Reed, 1966; Miller and Smith, 1970; Smith, 1980). The interaction of many hemolytic compounds with hemoglobin results in the generation of radicals which cause peroxidation of cellular membrane constituents (Beutler, 1969; Weed and Reed, 1966; MiIler and Smith, 1970). Such events, leading to erythrocyte damage, are often as-

HEMATOTOXICITY

OF CYCLOHEXANONE

125

OXIME

TABLE 5 HEMATOLOGICAL

VALUES OF CXLOHEXANONE AFTER INITIAL

0x1~ (CHO~TREATED AND CONTROL DOSING (RECOVERY GROUPS)

RATS 28 DAYS

Treatment (5x/week/2 weeks) Females

M&S

Distilled water Parameter

Units

Erythrocytes Hematocrit Total hemoglobin Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concn. Poikilocytosis Anisocytosis Polychromasia Howell-Jolly bodies Reticulocytes Circulating nucleated etirocytes Leukocytes Platelets

106/mm3

(20ml/h

PO)

CHO (300 w/h

Distilled water PO)

(20 a%

PO)

PO)

9.38 + 0.177’ 54.7 f 0.81 16.0 + 0.17

8.56 + 0.062’ 51.6 k 0.79 16.7 f 0.21 b

55 f 0.5

60 zk I.lb

17.0 + 0.32

19.6 + 0.34b

18.4 f 0.43

20.2 + 0.4Sb

32.5 + 0.54b 0.2

Scored scared smref %

30.9 + 0.47 0.6’ 0.8 1.4 0.0 0.7 + 0.13

31.4 + 0.79 1.2 1.00 1.4 0.0 I.0 f 0.37

32.5 k 0.49’ 0.2’ 0.80 1.0 0.6 1.0 k 0.24

Id/mm3 103/mm3 @/mm’

0.04 + 0.055 5.7 + 0.54 714 + 38.0

0.01 k 0.022

0.01 * 0.019 4.1 + 0.47 679 + 26.8

0.02 + 0.026 3.9 + 1.02 650 + 50.0

96 SCOR”

1.00

2.0 0.0 1.8 k 0.43’ 5.6 + 0.82 694 f 45

8.43 A 0.197 49.4 + 1.26 15.5 f 0.53

CHO (300 u&s

58

* 0.0

8.00 A 0.423 49.80 zk 2.45 16.20 + 0.73 61 f 0.8b

‘Mean+lSD. b Sign*cantly different from control, p < 0.0 1. c Siificantly different from control, p < 0.05. dO = parameter not present; 1 = minimally present; 2 = slightly present; 3 = moderately present; 4 = markedly present. c Nonapplicable. ‘0 = no cells containing parameter present; 1 = l-2 cells/l00 WBC; 2 = 3-5 cells/l00 WBC, 3 = 6-8 cells/100 WW; 4 = >8 cells/100 WEE.

sociated with elevated levels of methemoglobin. In the present study, CHO, administered orally to rats five times a week for a period of 2 weeks at dose levels ranging from 10 to 300 mg/kg, caused a dose-related reduction in the number of circulating erythrocytes. This was accompanied by hepatomegaly, splenomegaly, an increased degree of poikilocytosis, and elevated methemoglobin levels. Histopathological examination of the spleen revealed macrophages ladened with hemosiderin pigment. These results suggest that

subacute administration of CHO produced a hemolytic anemia, most likely resulting from CHO-induced oxidative damage to circulating erythrocytes. The failure to detect elevated levels of free serum hemoglobin, urine hemoglobin, or urine urobilinogen further indicates that the hemolysis occurred extravascularly and at a moderate rate. However, it should be kept in mind that 2 days elapsed between the administration of the final CHO dose and clinical analysis, making an interpretation of these clinical values less than definitive.

126

DERELANKO ET AL. TABLE6

CLINICAL VALUES OF MALE AND FEMALE CY~LOHEXANONE OXIME (CHOFTREATED AND CONTROL RATS 28 DAYS AFTER INITIAL DOSING (RECOVERY GROUPS) Treatment (SX/week/Z weeks) Females

M&S Distilled water Parameter

Units

Methemoglobin Femoral marrow myeloid/ erythroid ratio Glucose&phosphate dehydrogenase (erythrocyte)

CHO

(2Omlbis1-M (~w/kpo)

CHO

(3~w/bpo) 0.9 -+

0.8 f 0.26'

0.7 + 0.28

0.9 + 0.08

1.24 +_0.138

1.61 +- 0.294*

1.04 f 0.143

1.37 f 0.146’

18.47 + 1.609

23.17+ 0.960'

16.24k 0.433

19.60 + 0.963’

96

Units/gHb

DistiIIed water

(2OmlFg,po)

0.11

“Mean+ 1 SD. * Significantly difI&ent from control, p < 0.05. c Significantly different from control, p -c 0.0 1.

The increased degree of retictiocytosis, the presence of elevated numbers of circulating immature erythrocytes, and the erythroid hyperplasia of the bone marrow and spleen indicate that compensatory erythropoietic TABLE7 SPLEEN AND LIVER OXIME (CHO)-TREATED DAYS

AFTER

INITIAL

WEIGHTS OF CYCLOHEXANONE AND CONTROL ANIMALS

28

DOSING Treatment (SX/Week/2 weeks) Distilled

water

Units

(20ml/k&

g mp/g g

0.57 1.89 10.2 1 34.13

* + + k

0.050” 0.073 0.945 I.146

0.37 2.07 5.57 3 I .02

f + + +

0.029 0.170 0.289 0.827

PO)

(300

CHO mfk

PO)

Males

Spleen weight Spleen ratio’ Liver weight Liver ratio

mp/g

0.65 2.20 10.93 36.81

f 0.036b +0.132d + 0.828 + 1.858*

0.41 2.23 6.01 32.82

+ 0.029 AZ0.128 f 0.338 f 1.4286

Females Spleen weight Spleen ratio Liver weight Liver ratio

g me/z5 g me/g

“Mean+ISD. * Significantly different fmm control, p c 0.05. ‘Organ weighvbody weight ratio (relative weight). ’ Significantly ditkrent from mntrol, p +z 0.01.

mechanisms were fully functional in response to the decreasing erythroid mass. Moreover, the return to normal or near-normal hematological values in CHO-treated animals 14 days subsequent to termination of dosing suggests that the hemolytic effect of CHO is transient and complete recovery would probably occur upon cessation of exposure to CHO. Our findings are in good agreement with previous reports on CHO toxicity. Lomonova (1966) reported a decrease in total hemoglobin and erythrocyte number in rabbits exposedtoaO.l-to 1.0~g/kgoraldoseofCHO. Lomonova found a 20 to 509b elevation in methemoglobin 24 hr postdosing. The methemoglobin levels declined by the third day of the study. Tsulaya et al. (1975) reported decmased erythrocyte numbers and an elevation in methemoglobin in the blood of rats exposed to CHO via inhalation at dose levels ranging from 0.1 to 1.0 mg/m’ for a period of 6 to 10 weeks. The above effects were noted at the high dose level during the eighth week of the study. In contrast to our findings, these workers did not observe any compoundrelated effects in either the weight or histo pathology of the spleen or liver. Bone marrow

HEMATOTOXICITY

OF

CYCLOHEXANONE

OXIME

127

was not examined. A slight reduction in toxic effect on the erythrocyte, individuals blood catalase was also reported. This did deficient in various erythrocytic enzymes, not occur in our study. such as glucosed-phosphate dehydrogenase, Cyclohexanone has been reported to cause might be particularly sensitive to CHO. hematological effects similar to the oxime. REFERENCES (Koeferl et al., 1981). Cyclohexanone, inBAUER, J. D., ACKERMANN, P. G., AND TORO, G. jected iv into dogs at a dose level of 284 mg/ (1968). Bray’s Clinical Laboratory Methods, pp. 164kg for 18 to 2 1 days, produced a hemolytic 165. Mosby, St. Louis. anemia, with bone marrow hyperplasia, ex- BEUTLER, E. (1969). Drug-induced hemolytic anemia. tramedullary hematopoiesis, hepatomegaly, Pharmacol. Rev. 21,73-103. and hemosiderin deposition in the liver and FRUHMAN, G. J., AND GORDON, A. S. (1955). Quantitative effects of corticosterone on rat bone marrow. spleen. Proc. Sot. Exp. Biol. Med. 88, 130-131. Pokorny (1952) reported paralysis of the GAD, S. C. (1982). A neuromuscular screen for use in rear extremities of rabbits following iv adindustrial toxicology. J. Toxicol. Environ. Health 9, ministration of CHO at doses ranging from 691-704. 39 to 97 mg/kg. This was preceded by a loss GAD, S. C., AND WEIL, C. S. (1982). Statistics for toxicologists in Principles and Methods of Toxicology. of consciousness immediately following dos(A. Wallace Hayes, ed.), pp. 273-319. Raven Press, ing. No such neurobehavioral effects were New York. observed in the present study. KOEFERL, M. T., MILLER, T. R., FISHER,J. D., MART~S, Our data indicate that the hemolytic action L., GARVIN, P. J., AND DORN-ER,J. L. (1981). Itdluence of concentration and rate of intravenous administmtion of CHO may be greater in males than females; on the toxicity of cyclohexanone in beagle dogs. however, this interpretation may be misleadToxicol Appl. Pharmacol. 59, 2 15-229. ing. Hematological values of female control LOBUE, J., DORNFEST, B. S., GORDON, A. S., HURST, rats for the dosing phase of the study were J., AND QUASTLER, H. (1963). Marrow distribution in noticeably lower than is common for this rat femurs determined by cell enumeration and ‘Te labeling. Proc. Sot. Exp. Biol. Med. 112,1058-1062. strain of rat in our laboratory (compare the LOMONOVA, G. V. (1966). Changes in the blood of values from this group to those of the female rabbits under the action of cyclohexanone oxime. Gig. control group from the recovery phase of the Tr. ProJ: Zabol. 10,58-70. study). This may explain the statistically sig- MILLER, A., AND SMITH, H. C. (1970). The intracellular nificant elevation in hematological values, and membrane effects of oxidant agents on normal red cells. Brit. J. Haematol. 19, 417-428. compared to controls, of the female group which received CHO at a dose level of 10 POKORNY, F. (1952). Toxicological trials with cyclohexanone oxime, ccaprolactam, and e-aminocaproic acid: mg/kg. The mason for the depressed hemaBiological cross comparison of these compounds. S. tological values of the female controls is Lek. 54, 2847. unknown. Therefore, while it is possible that SMITH, R. P. (1980). Toxic responses of the blood. In Toxicology: The Basic Science of Poisons (J. Doull, the degree of hemolysis induced by CHO C. D. Klaassen, and M. 0. Amdur, cds.), 2nd ed. pp. may have been greater in males than females, 3 1 l-33 I. Macmillan, New York. a definitive statement regarding a sex differ- TARLOV, A. R., AND KELLERMEYER, R. W. (1961). The ence in CHO toxicity must await further hemolytic effect of ptimaquine. XI. Decreased catalase testing. activity in primaquine-sensitive erythrccytes. J. Lab. Clin. Med. 58, 204-2 16. In conclusion, the results of the present TSULAYA, V. R., PAREVERZEVA,E. A., AND ARSEN’EVA, study show that CHO causes a hemolytic S. S. ( 1975). Hygenic standardization of cyclohexanone anemia in the rat following subacute oral oxime in the atmosphere. Gig. Sanit. 2, 23-25. exposure. The hemolysis appears to result WEED, R. I.. AND REED, C. F. (1966). Membrane from CHO-induced oxidative damage to the alterations leading to red cell destruction. Amer. J. Med. 41, 681-698. erythrocyte. Because of the nature of the