International Archivesof
Oeeupationald
Int Arch Occup Environ Health (1988) 60:7-14
Enironmental Health
© Springer-Verlag 1988
Leukocytosis and low serum Ig A in workers exposed to the epoxy compound, t-methyl-3-phenylglycidate Yuichi Yamada, Teruhiko Kido, Masao Ishizaki, Ryumon Honda, Ikiko Tsuritani, Haruki Yamaya, and K6ji Nogawa Department of Hygiene, Kanazawa Medical University, Uchinada, Ishikawa 920-02, Japan
Summary Forty-nine out of 54 male workers engaged in the production of an epoxy compound, tmethyl-3-phenylglycidate, showed skin symptoms in varing degrees that may be due to the skin-irritative effect of the compound The exposed workers were also shown to have subjective symptoms which may be related to the irritative property of the compound on surface tissue Laboratory examinations on the blood obtained from the exposed workers showed significantly higher values of leukocyte concentration as compared with the non-exposed controls This was chiefly caused by the increase of neutrophilic granulocytes and T-cell lymphocytes Serum Ig A levels of the exposed workers were shown to be significantly lower than those of the control group Hemoglobin concentration, hematocrit value and red cell count of the exposed workers remained at the same level as those of the control subjects Liver or kidney damage was not found in biochemical analyses on the sera of exposed workers. Key words: Dermatitis IgA Epoxy compound
Leukocytosis Low serum t-Methyl-3-phenylglycidate
the chemical plant consulted us for countermeasures. In the preliminary health examination, 42 out of the 49 workers, who had been engaged in the process and must have been exposed to the epoxy compound, showed skin symptoms in varing degrees Besides these skin symptoms, many of the workers were found to have high white blood cell concentrations in their peripheral blood, exceeding 10000 per cubic mm in some cases. Some of epoxy compounds are known to be toxic to the liver or kidney of animals l2, 10, 24 l The effects on hematopoietic systems in peripheral blood or in bone marrow have also been observed in animals exposed to epoxy compounds l7, 11, 13l However, these systemic effects of epoxy compounds on human beings has not yet been fully confirmed l8 l Thus, the well-designed health examinations on the workers in the chemical plant were carried out six months after the outbreak of dermatitis in order to confirm the hematological changes observed in the preliminary study and to find other systemic effects of the epoxy compound in the exposed workers The results of the health examinations are presented in this paper. Materials and methods
Introduction The most frequently observed health hazard in people exposed to epoxy compounds is contact dermatitis, which may be due to either primary irritation or allergic sensitization by the compounds l8 l The acute outbreak of contact dermatitis was found in workers exposed to an epoxy compound, t-methyl-3-phenylglycidate, in a chemical plant The management of Offprint requests to: Y Yamada (at the above adress)
The epoxy compound, t-methyl-3-phenylglycidate, is produced as an intermediate substance in the production of a newly developed drug, Diltiazem, for cardiovascular diseases The drug acts as a calcium antagonistic agent in vascular smooth muscles l1, 6l A brief outline of the production process is shown in Fig 1 Two liquid materials, aldehyde and chloroacetate, are combined by the so-called Darzen's reaction l22 l The epoxy compound produced is crystalline It is collected by centrifugation and then dried and crushed into a powder The workers had been involved in pouring the raw material liquids into a reaction tank and in conveying the produced epoxy compound to the crushing equipment.
Y Yamada et al : Leukocytosis and low serum IgA by exposure to an epoxy compound
8 Materials
/0\ HC -CH
CHO ClCH 2COOCH
3
OCH OCH 3 P Anisaldehyde
Methyl chloroacetate (liquid )
(liquid)
COOCH,
f
Na-OCH 3
t
OCH 3 Methyl-3 phenyl glycidate ( crystalline)
Fig 1 Materials and the production process of the epoxy compound, t-methyl-3-phenylglycidate The workers had been involved in pouring the raw material liquids into a reaction tank and in conveying the produced epoxy compound to the crushing equipment
Process-
Synthetic
reaction
Centrifugation
Drying
The number of workers who had been engaged in the process was 57 at the time of the examinations, fifty-four were men and three were women In this paper, however, only the results of the examinations on the 54 male workers are reported Age-matched control subjects were chosen among male workers in the same plant, who had not been involved in the process. All subjects, both the exposed and the control, were asked to gather at an examination facility close to their factory by 8:00 h one morning They were to refrain from eating, drinking, smoking or doing any physical exercise after awaking that morning Blood samples were collected from 9:00 to 10:00 h that morning. The form of the examinations was as follows: questionnaires about subjective symptoms, past experience and familial histories of major allergic diseases, such as bronchial asthma, nasal allergy, allergy to drugs, atopic dermatitis, repetitious urticaria, contact dermatitis; presence or absence of skin symptoms and the lapsed time from the exposure to the epoxy compound to the appearance of the symptoms; observations on the skin lesion and blood analyses. The right median cubital vein was punctured using a disposable Venoject needle (Termo, Japan), and 3 ml of blood was aspirated into a vacuum glass tube with EDTA-3K (Venoject, Termo) for hematological analyses and 7ml of blood was aspirated into a plain vacuum glass tube (Venoject, Termo) for biochemical and serological analyses on the serum The left median cubital vein was punctured by a heparinized disposable polypropylene syringe (Termo) to collect blood to examine the lymphocyte subpopulation and lymphocyte blastoid transformation The blood samples (kept at O°C) were transported to our laboratory immediately after collection None of the laboratory technicians were given any information concerning the exposure to the epoxy compound They carried out the analyses as part of their routine work. Hemoglobin concentration, hematocrit value, red cell count and white cell count were measured by an automatic analyzer, THMS, H-6000 (Technicon) Two blood smear slides of each subject were prepared from the blood obtained for the hematological analyses Differential counting of the white blood cells on the smear slides was done by an experienced hematologist, who counted 200 cells in each smear slide. Serum was analyzed biochemically by an automatic analyzer, SMAC III (Technicon) for total protein, bilirubin, GOT, GPT, y-GTP, Al-P, LDH, BUN, creatinine, etc The concentrations of Ig G, IgM and Ig A in the serum were measured by a radioimmunoassay l 15 l, and Ig E was measured by a radio-
immunosorbent assay (RIST, Phadebas) l 14 l RA test, LE test and anti-nuclear antibodies were also analyzed in the serum. The lymphocytes for the immunological examinations were obtained by a differential centrifugation method with FicollConray solution (d = 1 077) l21 l T-cell lymphocytes were determined by E-rosette formation on sheep erythrocytes treated with neuraminidase l19 l B-cell lymphocytes were determined by a membrane immunofluorescence technique for detecting cell-surface immunoglobulin l 12 l The lymphocyte blastoid transformation test was performed according to the method described by Takiguchi et al l 20l, using a semi-automatic multiple sample harvester (Otto-Hiller) The lymphocytes were incubated for four days in Microtest II Tissue Culture Plate # 3040 (Falcon) with and without a mitogen, phytohemaggulutinin (PHA-P, Difco) or concanavalin A (Con A, x2, Miles-Yeta) The incubating solution was made with RPM 1640 (Nissui), NaHCO 3, fetal calf serum (GIBCO) 3 Hthymidine was added on the last incubation day and incorporation into the lymphocytes was measured by a scintillation counter (Beckman).
Results Age distribution of the 54 male workers and the duration that they had been involved in the process are summarized in Table 1 About half of the workers had been engaged for longer than one month All six workers who had been engaged for fewer than three days had suffered from severe dermatitis as soon as they had become involved in the process, and therefore had been removed from the process before the time of examination. Forty-nine of the 54 male workers had recognized some skin symptoms, such as itching, redness of the skin and an eczema-like rash None of them, however, showed overt skin lesions at the time of the examination because improvement in the working conditions had been enacted The appearances of skin symptoms after being involved in the process are summarized in Table 2 Forty-two of the 49 workers had recognized skin symptoms within three days after
9
Y Yamada et al : Leukocytosis and low serum IgA by exposure to an epoxy compound
they had become involved in the process for the first time. The 49 workers were devided into two groups according to the severity of skin symptoms Thirty workers who had had an eczema-like rash were clasTable 1 Age distribution and the duration that workers had been engaged in the production process Duration
1 or 2 d 2 or 3 d About 1 week 2 or 3 weeks More than 1month More than 3 months
Age (years) 20-29 30-39 (n= 11) (n = 9) 1
40-49 50 Total (n=23) (n= 11) (n=54)
1
2
1 2 4
2
4 2 7
2
2
4
2
10
1
1
1
1
4
6
4
11
6
27
Table 2 Lapsed time from the engagement in the production process to the appearance of skin symptoms Lapsed time Age (years)
lor 2d 2 or 3 d About 1 week 2 or 3 weeks More than 1 month Uncertain
20-29 30-39 (n=11) (n= 9)
40-49 (n = 23)
50 Total (n=11) (n= 54)
7 2
11 7
5 3
5 2 2
1 1
28 14 2 1
2
1
3 1
sified as the "severe dermatitis" group, 19 who had shown only redness of the skin or had experienced itching without overt skin lesions were classified as the "mild dermatitis" group The five other workers who had not shown any skin symptoms were called the "no dermatitis" group. Past experience and hereditary tendencies of major allergic diseases were compared between the exposed workers and the control subjects, and among the three exposed groups However, no significant differences were observed between the exposed and the non-exposed subjects The remarkable finding was that none of the five workers in the "no dermatitis" group had had major allergic diseases in the past. As shown in Table 3, the exposed workers proved to have seven items of subjective symptoms more frequently than the control group: general fatigue, lowering of visual acuity, nasal obstruction, nasal discharge, susceptibility to common colds, expectoration and a feeling of heartburn The differences were significant in the lowering of visual acuity and in the expectoration These symptoms, however, were not correlated with the severity of skin symptoms in the exposed workers. In the results of the hematological studies, there were no differences of hemoglobin concentration, hematocrit value and red cell count in peripheral blood between the exposed and the non-exposed subjects However, white cell counts of the exposed subjects were found to be significantly higher than those of the control subjects The results of the hemoglobin concentration and the white cell count are illustrated in Fig 2 and in Fig 3, respectively. The concentrations of neutrophilic granulocytes, eosinophilic granulocytes and lymphocytes in peripheral blood were calculated from total white cell concentration and the percentages of those cells in the blood smear slides The results are illustrated in
Table 3 Subjective symptoms of the exposed workers according to the severity of the skin lesions and comparison with the nonexposed controls Symptoms
General fatigue Lowering of visual acuity Nasal obstruction Nasal discharge Susceptibility to colds Expectoration Feeling of heart-burn
Exposed
Nonexposed
Severe dermatitis ln = 30 (%)l
Mild dermatitis ln = 19 (%)l
No dermatitis ln = 5 (%)l
ln = 17 (%)l
5 (16 7) 9 (30 0) 2 (6 7) 5 (16 7) 7 (23 3) 8 (26 7) 6 (20 0)
2 (10 0) 6 (31 6) 4 (21 1) 2 (10 5) 5 (26 3) 3 (15 8) 3 (5 8)
0 1 (20 0) 1 (20 0) 0 1 (20 0) 1 (20 0) 0
01 ( 5 9)* 002 (11 8) 0 -* 1 (5 9)
* The difference is significant (P < 0 05) between the exposed and the non-exposed subjects in Fisher's direct probability analysis
Y Yamada et al : Leukocytosis and low serum IgA by exposure to an epoxy compound
10 (g/di) 20 19 18 c
.E
To
17 16 15 14 13
E I
Fig 2 Hemoglobin concentration in peripheral blood The exposed workers are shown as solid circles and the non-exposed controls as open circles There is no significant difference between the exposed and the non-exposed subjects
12 11 10
20 -29
30 -39
40
Age
49
50
group
13
12
11 0
I I
10
9
V
l
0 I
8
i
3. ..
7
6
4 3 20-29
40-49
30-39 Age
50-
Fig 3 White blood cells concentration in peripheral blood Solid circles are exposed workers and open circles are control subjects The WBC concentrations of exposed workers are significantly higher than those of the control subjects in all age groups except for the twenties.
group
Fig 4 The difference of the concentration of neutrophilic cells between the exposed and the non-exposed subjects was highly significant (P<0 01) The concentration of lymphocytes was also somewhat higher in the exposed subjects, but the differences was not statistically significant ( 0 05 _ P < O 10). Biochemical analyses on serum did not show any differences between the exposed and the control subjects, although one control subject was found to have a chronic liver disease, probably chronic non-A, nonB hepatitis.
Immunological studies were carried out on 23 exposed subjects and nine non-exposed controls, randomly selected from each group Two exposed subjects were excluded from the following discussion because they showed extremely high Ig E levels in the sera, and they had had bronchial asthma in their childhood One control subject was also excluded because of liver damage found in the biochemical analyses on blood. The means and standard deviations of serum Ig G, Ig M, Ig A and Ig E concentrations are shown in Table
11
Y Yamada et al : Leukocytosis and low serum Ig A by exposure to an epoxy compound (xlO/mm,'j
3
x o10/mm ) 10
(xlO Ymm) 10
I1
9
9
9
o
8
8
8
I .E z
7
7
7
6
6
5
5
4
4
o
I ii
r
6
== c
5
L
4
t
i
4r u
3
3
3
t
I.
Fig 4 Concentration of neutrophilic granulocytes, eosinophilic granulocytes and lymphocytes in peripheral blood Solid circles are exposed and open circles are control subjects. The neutrophilic granulocyte concentation is significantly higher (P < 0 05) and the lymphocyte concentration is somewhat higher (0 05 _ P < O 10) in the exposed workers as compared with the controls
8
ro
2
2
.
2
r
1
J
&
r
1
'
O 0
0
Neutrophilic granulocyte
Lymphocyte
Eosinophilic granu locyte
Table 4 The levels of serum immunoglobulins in the exposed workers and in the non-exposed controls M: mean Subjects
N
Age (years) M+SD
IgG (mg/dl) M+SD
Ig M (mg/dl) M+SD
IgA (mg/dl) M±SD
Ig E (IU/ml) M±SD
Exposed Non-exposed
21 8
40 1 + 10 2 37 3 ± 8 2
1270 ± 302 1395 ± 352
97 4 + 33 6 84 4 + 18 5
188 + 69 * 261 ± 73
158 + 146 255 ± 174
* The difference is significant (P < 0 05) between the exposed and the non-exposed subjects in Student's t-test
x 10 ' 10
a 0I
x 10
x 10
5
10
10
9
9
9
8
8
8
7
7
7
6
6
I
E E
6 I.
0
5
6
ao -o l i. ;x
I
5 4
4
r
L
5 4
3
3
2
2
1
1
1
0
0
3
S.
2
I
C :
l I E
ControlI
0 PH (+)
Con Ai +f)
Fig 5 Results of lymphocytes blastoid transformation test by common mitogens: PHA and Co A. Solid circles are exposed workers and open circles are controls The exposed subjects showed 3 significantly higher incorporation of H-thymidine into lymphocytes without mitogen (in control study) than the controls, but there are no significant differences between the two groups after the incubations with mitogens lin PHA (+) and Co A (+) studiesl
12
Y Yamada et al : Leukocytosis and low serum IgA by exposure to an epoxy compound
4 The concentrations of IgG and IgM were not different between the exposed subjects and the non-exposed subjects Ig A and Ig E levels were somewhat lower in the exposed subjects The difference was significant in the Ig A concentration (P < O 05) The results of analysis on lymphocyte subpopulation, T-cell and B-cell, and of the blastoid transformation test induced by PHA or Con A were summarized as follows The concentrations of T-cell and B-cell lymphocytes were calculated from the percentages of these cells in total lymphocytes and the concentration of the total lymphocytes which was obtained from the white blood cell count and blood smear examination as mentioned above The concentrations of T-cell and B-cell were both somewhat higher in the exposed subjects The difference was more marked in T-cell lymphocytes ( 0 05 < P < 0 10). In the blastoid transformation test, 3 H-thymidine incorporation into the lymphocytes incubated without mitogen was significantly higher in the exposed subjects After incubation with PHA, the lymphocyte 3 H-thymidine uptake of the exposed subjects was somewhat lower than those of the controls although the difference was not significant (O 05 P < O 10). In the case of incubation with ConA, however, 3Hthymidine uptake of lymphocytes in the exposed subjects was almost equal to that in the controls These findings are illustrated in Fig 5. The exposed workers and the control subjects were also examined for RA test, LE test and antinuclear antibodies in the sera, but no positive results were found either in the exposed or in the non-exposed subjects.
Discussion Forty-nine out of 54 male workers involved in the production of the epoxy compound, t-methyl-3-phenylglycidate, showed skin symptoms in varing degrees. Forty-two of the 49 workers had recognized the skin symptoms within three days after they had become involved in the process for the first time Thus, the skin symptoms were considered to be caused chiefly by the direct irritative effect of the compound, but the sensitization to the compound could not be denied in some workers who had recognized the skin symptoms more than one month after the exposure. The exposed subjects showed significantly higher incidence of the subjective symptoms, such as a lowering of visual acuity and expectoration as compared with the non-exposed controls These subjective symptoms were also considered to be caused by the direct irritative effect of the compound upon the mucous membrane l8 l These symptoms did not cor-
relate with the severity of the skin symptoms of the exposed workers. In laboratory analyses for hematological, biochemical, serological and immunological effects of the epoxy compound, white cell count in peripheral blood and 3 H-thymidine uptake of the lymphocytes incubated without mitogen were significantly higher, but serum Ig A level was significantly lower in the exposed workers as compared with the controls In the elevation of white cell count, the chief component of the white cells was neutrophilic granulocytes, and Tcell lymphocytes were also increased to a mild degree. As far as leukocytosis is concerned, many physiological and pathological factors should be taken into account, since the concentration of white blood cells in peripheral blood is easily fluctuated by those factors Among the pathological factors, inflammation, acute or chronic, due to biological or chemo-physical agents is the most common and strongest cause l23l for the increase of white blood cells None of the subjects, except a single exposed one who had a mild cold, had any inflammatory diseases, such as tonsilitis, skin abcess or urinary tract infection, at the time of the examination Physiological factors, such as digestion, physical exercise and diurnal variations, are known or suspected to cause the elevation of leukocyte concentration l23 l Since so many factors could have affected the white cell count in the peripheral blood, the examinations, were designed very carefully in order to make the physiological conditions of the subjects as similar as possible Thus, we thought we could avoid the acute influences caused by physiological factors on the white cell count of the subjects. Cigarette smoking is suspected to cause an increase of leukocytes in blood chronically l5 l Thus, cigarette smoking and alcohol consumption were compared between the exposed and the non-exposed groups Smokers were found in 40 of the 54 male exposed subjects (74 1 %) and in 13 of the 17 controls ( 76 5%) Of the subjects who drink alcohol more than three times a week, 32 were found in the exposed group ( 59 3%) and eight in the control group (47 1 %) The differences of the percentages of smokers and drinkers between the two groups were not significant. The biases of the examiners were also avoided in the examinations by using automatic equipment and by the blind analyses From these basic considerations, we can conclude that the increase of white blood cells in the peripheral blood of the workers exposed to the epoxy compound, found in the preliminary study six months before, was confirmed in the present examination.
Y Yamada et al : Leukocytosis and low serum Ig A by exposure to an epoxy compound
The dermatitis caused by the epoxy compound is an inflammation Thus, it might be possible to assume that the elevation of white blood cell count observed in the exposed subjects was not caused by the direct effect of the compound on hematopoietic system, but rather was as a caused consequence of the skin inflammation induced by the compound In the exposed workers, however, the concentration of white cells in blood did not correlate with the severity of skin lesions Therefore, we think the observed elevation of white blood cell count does not merely reflect the dermatitis on the skin of workers. Although there is no report about hematological changes occurring in people exposed to epoxy compounds, except for ethylene oxide l3 l, some animal experimental studies have shown hematological effects caused by various kinds of epoxy compounds l3, 8 l In many cases, the animals showed a depression of leukocyte concentration, both polymorphonuclear cells and lymphocytes, after exposure to the compounds The decrease of hemoglobin content in blood has sometimes been observed In human studies, there are some reports l3l about ethylene oxide exposure, in which lymphocytosis was commonly observed One of these reports showed the decrease of hemoglobin content in blood These findings in animal and human studies seem to differ from the results of the present study, where the leukocytes, especially neutrophilic granulocytes, increased in the concentration in blood, whereas the hemoglobin content remained in the normal range Epoxy compounds are alkylating agents, which are known to act as "radiomimetic" substances, and low level irradiation is known to induce the increase of leukocytes, both lymphocytes and polymorphonuclears, in the peripheral blood l4 l One animal study also showed the elevation of leukocyte concentration in the peripheral blood l8l Thus, we think the leukocytosis found in the present examination does not conflict with previous reports The finding of leukocytosis in workers exposed to t-methyl-3-phenylglycidate is the first report about the hematological changes occurring in humans exposed to an epoxy compound, except for ethylene oxide. Immunological observations on the workers showed significantly lower levels of serum IgA and the significantly higher incorporation of 3 H-thymidine into lymphocytes without stimulation by mitogen as compared with the control subjects The meaning of the low serum IgA in exposed workers is not clear, because the Ig A and Ig E levels in the exposed workers were lower than in the controls, but the Ig G and Ig M levels of the exposed workers were shown to remain at the same level as those of the controls Further, the biological role of IgA in serum has not yet
13
been clarified, although the secretory Ig A on the mucous membrane surface is well known as playing an important role in the local defence mechanism against biological agents However, there are some reports on the decreased serum Ig A level induced by chemicals The chemicals are the drugs used in treatments of rheumatoid arthritis or epilepsy They are D-penicillamine l 9, 18 l or phenytoin l16, 17l These drugs can induce selective suppression of Ig A in the sera of the patients Therefore, there is a possibility that the low serum Ig A observed in workers in the present study has a relation to the exposure to the compound The increased 3 H-thymidine incorporation into lymphocytes of the exposed workers without stimulation by mitogen may simply reflect the proliferation of lymphocytes in blood of the exposed subjects, but it might also have a relation to the decreased serum Ig A In any case, the immunological alterations induced by epoxy compounds have not been documented in both animal experimental and human epidemiological studies Thus, it should be further investigated. In biochemical analyses on the sera of exposed workers, the other systemic effects of the epoxy compound were not found either in liver function tests, such as GOT, GPT, y-GPT, LDH, Al-p, or in renal function tests such as BUN, creatinine.
References 1 Breeman C, Mangel A, Fahim M, Meisheri K (1982) Selectivity of calcium antagonistic action in vascular smooth muscle Am J Cardiol 49:507-510 2 Dobryszyeka W, Warwas M, Woytofi A, Woytoti J, Szacki J (1974) Serum proteins and enzymes in pregnant rats treated with epoxy resin or triethylene-tetraamine stabilizer Arch Toxicol 33: 73-80 3 Ehrenberg L, Hussain S (1981) Genetic toxicity of some important epoxides Mut Res 86:1-113 4 Elson LA (1963) Radiation and radiomimetic chemicals. Butterworths, London, pp 49-60 5 Friedman GD, Siegelaub AB, Seltzer CC, Feldman R, Collen MF (1973) Smoking habits and the leukocyte count. Arch Environ Health 26:137-143 6 Fujiwara S, Ito Y, Itoh T, Kuriyama H, Suzuki H (1982) Diltiazem-induced vasodilatation of smooth muscle cells of the canine basilar artery Br J Pharmacol 75: 455-467 7 Hine CH, Kodama JK, Guzman RJ, Dunlap MK, Lima R, Loquvam GS (1961) Effects of diglycidyl ether on blood of animals Arch Environ Health 2: 37-50 8 Hine CH, Rowe VK (1981) Epoxy compounds In: Clayton GD, Clayton FE (ed) Patty's industrial hygiene and toxicology, 3rd edn John Willey & Sons, New York, pp 2141-2257 9 Hjalmarson O, Hanson LA, Nilsson LA (1977) IgA deficiency during D-penicillamine treatment Br Med J 1: 549549 10 Hollingsworth RL, Row VK, Oyen F, McCollister DD, Spencer HC (1956) Toxicity of ethylene oxide determined
14
11 12 13 14
15
16 17
18
Y Yamada et al : Leukocytosis and low serum Ig A by exposure to an epoxy compound on experimental animals AMA Arch Ind Health 13:217227 Jacobson KH, Hackley EB, Feinsilver L (1956) The toxicity of inhaled ethylene oxide and propylene oxide vapors. AMA Arch Ind Health 13:237-244 Kawamura A, Murata M (1976) The detection of lymphocyte-surface-immunoglobulin Rinsho Meneki lClin Immunoll 8:689-695 (in Japanese) Kodama JK, Guzman RJ, Dunlap MK, Loquvam GS, Lima R, Hine CH (1961) Some effects of epoxy compounds on the blood Arch Environ Health 2:56-67 Miyamoto A (1974) The measurement of Ig E by RIST In: Migita S (ed) Methods in immunological experiment B. Japanese Society for Immunology, Kanazawa, Japan, pp 1057-1060 (in Japanese) Nishizawa Y, Kishimoto T (1975) The measurement of anti-DNP and total Ig G, Ig A, Ig M by radioimmunoassay. In: Migita S (ed) Methods in immunological experiment B. Japanese Society for Immunology, Kanazawa, Japan, pp 970-974 (in Japanese) Seager J, Jamison DL, Wilson J, Hayward AR, Soothill JF (1975) IgA deficiency, epilepsy, and phenytoin treatment. Lancet 2:632-635 Sorrell TC, Forbes IJ, Burness FR, Rischbieth RHC (1971) Depression of immunological function in patients treated with phenytoin sodium (sodium diphenylhydantoin). Lancet 2: 1233-1235 Stanworth DR, Johns P, Williamson N, Shadforth M, Felix-Davies D, Thompson R (1977) Drug-induced IgA deficiency in rheumatoid arthritis Lancet 1:1001-1002
19 Tachibana T, Yoshida A (1974) Microanalysis of human T cell and B cell In: Migita S (ed) Methods in immunological experiment A Japanese Society for Immunology, Kanazawa, Japan, pp 455-462 (in Japanese) 20 Takiguchi T, Konaka Y, Shimizu S, Konda S, Shigehara M (1976) Human lymphocytes blastoid transformation test using a semi-automatic multiple sample harvester In: Migita S (ed) Methods in immunological experiment B. Japanese Society for Immunology, Kanazawa, Japan, pp 1426-1435 (in Japanese) 21 Tsuji K (1971) Separation of lymphocytes by a differential centrifugation method, Conray 400-Fincoll method In: Magita S (ed) Methods in immunological experiment A. Japanese Society for immunology, Kanazawa, Japan, pp 443-446 (in Japanese) 22 Windholz M, Budavari S, Stroumtsos Fertig MN (1976) The Merck Index, 9th edn Merck&Co , Rahway, NJ, USA 23 Wintrobe MM, Lee GR, Boggs DR, Bithell TC, Athen JW, Foerster J (ed)( 1974) Clinical hematology, 7th edn. Lea &Febiger, Philadelphia, pp 243-276 24 Woytofi J, Szacki J, Woytofi A, Warwas M, Dobryszycka W (1975) Tissue enzyme changes in pregnant rats treated topically with epoxyk resin (Epidian 5) or triethylenetetramine stabilizer Toxicol Appl Pharmacol 32:5-10
Received November 25, 1986 / Accepted June 30, 1987
