The Effect of Chronic Benzene Intoxication on the Phagocytic Activity of Leukocytes in Rabbits

A. P. Volkova

From the Department of General Hygiene, First Moscow Order of Lenin Medical Institute named after I. M. Sechenov

Reducing illness among industrial workers is one of the pressing tasks of Soviet public health. The relationship between the incidence of certain diseases and adverse working conditions is well established. Important data have been obtained, for example, on workers exposed to benzene at artificial leather factories in Leningrad. Among workers showing signs of chronic benzene intoxication, substantial losses of working time were attributed to influenza, tonsillitis, and pulmonary tuberculosis (O. N. Olimpieva). The effects of a number of chemicals—lead, chlorine, fluorine, carbon monoxide, and benzene—on immunobiological reactivity have been studied by a number of Soviet and foreign authors (Ya. D. Sakhnovsky, Sh. G. Perlina, L. L. Kandyba, G. A. Ionkin and M. N. Khanin, Ya. I. Melnik and R. D. Gabovich, V. K. Navrotsky, and others). Experimental studies by J. Hectoen showed that benzene intoxication reduced resistance to infection in experimental animals and caused dormant infections to flare up again. There is thus reason to believe that benzene intoxication reduces the body’s resistance to infection. However, this question remains insufficiently studied.

The present study investigates the effect of chronic benzene intoxication on the phagocytic activity of leukocytes in rabbits, since this activity is an important factor in the body’s defense against infection. The second stage of the work is a study of the phagocytic activity of blood from workers exposed to benzene.

The experimental studies consisted of four series of experiments. Rabbits were used because benzene relatively readily produces characteristic blood changes in them. A total of 28 animals were studied, eight of which served as controls. The benzene concentrations used for exposure were those most commonly encountered in industrial settings: 0.5, 0.2, 0.05, and 0.02 mg/L. Animals in each series were exposed by a dynamic method for 3 hours daily for 3 months. Throughout the experiment, the benzene concentration in the air of the exposure chamber was monitored. Benzene was measured by the method of T. N. Kozlyaeva and I. G. Vorokhobin.

Leukocyte phagocytic activity in the rabbits was determined by the generally accepted method of G. E. Platonov. A suspension containing 1.5 billion organisms from a 3-day culture of Bac. Fridman was used as the target for phagocytosis. To determine the phagocytic index, the number of phagocytosing cells per 100 neutrophils was counted in a smear. The number of microbes phagocytosed by a single neutrophil was also recorded (the phagocytic number). In addition to leukocyte phagocytic activity, the animals’ blood was examined for hemoglobin content and numbers of erythrocytes, leukocytes, platelets, and reticulocytes; differential leukocyte counts were also performed. The animals’ weight and behavior were monitored.

Before each successive series of experiments, baseline levels of all indicators were established.

Following three months of daily benzene exposure, the rabbits developed typical benzene intoxication. The most pronounced changes were observed in animals that inhaled benzene at a concentration of 0.5 mg/L. These consisted primarily of a reduction in leukocyte phagocytic activity, apparent as early as the third week of exposure. Whereas the animals’ mean phagocytic index was 53% before the experiment, it had fallen to 15% by the end of the thirteenth week of exposure (see figure). In individual rabbits, only 7 of 100 neutrophils were phagocytosing cells by the end of the experiment. The number of phagocytosed microbes also decreased: by the end of exposure, the mean number of microbes phagocytosed per neutrophil had fallen from 10 to 2–3. No changes in phagocytic activity were observed in the control animals.

At a concentration of 0.5 mg/L, benzene intoxication manifested as leukopenia, anemia, thrombocytopenia, and reticulocytosis. Thus, hemoglobin fell from 54 to 45%, the erythrocyte count from 4,500,000 to 54,000, while the reticulocyte count increased from 6.1 to 18%. The differential leukocyte count initially showed neutrophilia, followed by lymphocytosis and monocytopenia. Qualitative changes were expressed as transient anisocytosis.

The experimental animals lagged behind the controls in weight, and changes in behavior were observed: by the end of the experiment they became lethargic and inactive. In cases where the course of benzene intoxication was complicated by a purulent focus in the body (infected abrasions on the rabbits’ paws), despite the high benzene concentration, which in healthy rabbits caused

a picture of depressed hematopoiesis, infected animals exhibited leukocytosis reaching 15,000–17,000. At the same time, their hemoglobin levels and erythrocyte counts were relatively high. Leukopenia, characteristic of benzene intoxication, appeared only at the beginning of the ninth week of exposure. Infected animals, however, showed a greater decline in the phagocytic activity of leukocytes than healthy animals: by the end of exposure, 15 out of 100 neutrophils were phagocytic in healthy rabbits, compared with only 12 in infected rabbits. It may therefore be assumed that the presence of foci of purulent infection in an animal alters its response to benzene, making it atypical: leukocytosis may occur instead of leukopenia. Thus, inhalation of benzene at a concentration of

Figure labels: Baseline; start of exposure; exposure; recovery period; time in weeks; control; end of exposure. Concentrations: 0.5, 0.2, 0.05, and 0.02 mg/L.

Phagocytic activity of leukocytes (means for 28 rabbits).

0.5 mg/L causes typical benzene intoxication in animals, with marked suppression of leukocyte phagocytic activity; these changes begin before changes in the blood.

In the second series of experiments, at a benzene vapor concentration of 0.2 mg/L, similar changes in the phagocytic activity of blood leukocytes were observed. However, they began later and were less pronounced.

At a benzene concentration of 0.05 mg/L (the third experimental series), phagocytic activity, as in the first two series, underwent substantial changes. The phagocytic index decreased from 55% at the beginning of the experiment to 27% by the thirteenth week of exposure (see figure). The number of microbes phagocytosed by a single neutrophil also decreased, from 8–10 at the beginning of the experiment to 2–3 at the end. Changes in leukocyte phagocytic activity began in the fifth week of exposure and preceded blood changes, which manifested as moderate leukopenia, anemia, thrombocytopenia, and reticulocytosis. No changes in the differential leukocyte count were observed. The weights of the experimental animals did not differ from those of the controls.

In this series, the rates at which leukocyte phagocytic activity and the blood picture recovered after exposure ended were also examined.

The altered measures recovered in reverse order: leukocyte phagocytic function recovered only three months after exposure ended, whereas white blood cell measures, platelet counts, and reticulocyte counts returned to normal after approximately two months. Hemoglobin percentage and erythrocyte count recovered most rapidly.

Thus, at a benzene concentration of 0.05 mg/L, mild benzene intoxication occurs, accompanied by a decrease in leukocyte phagocytic activity. As in the first two series, blood changes began later than changes in leukocyte phagocytic activity.

Because exposure to benzene at a concentration corresponding to the maximum permissible level reduced leukocyte phagocytic activity, a fourth experimental series was conducted at a benzene concentration of 0.02 mg/L. In this series, phagocytic activity began to decline in the eighth week of exposure, and the phagocytic index decreased to 35% at the end of the twelfth week, compared with 52% at the beginning of the experiment. No notable blood changes were observed, apart from slight leukocytosis (10,000–11,000), which began in the sixth week of exposure and persisted throughout the experiment. Apparently, at this benzene concentration, a phase of stimulation of the hematopoietic organs occurred.

After the experiments, animals from all series were killed and their organs subjected to pathological examination. In animals of the first and second series

The following changes were found in the lungs: numerous pinpoint hemorrhages, merging in places into extensive hemorrhages measuring 3 × 4.5 mm; on sectioning the lung tissue, a bloody, frothy fluid flowed out. The liver was flabby, and its tissue tore easily under pressure, leaving ragged edges; the spleen was small, and its pulp was loosened. Pinpoint hemorrhages were present in the gastric mucosa, most often in the region of the greater curvature; numerous pinpoint hemorrhages were also present in the medulla and cortex of the kidneys. The bone marrow of the tibiae was paler than in the control animals. No notable changes in the organs were observed in rabbits of the third and fourth series.

Conclusions

1. Inhalation of benzene vapor at concentrations of 0.5, 0.2, 0.05, and 0.02 mg/L for 3 months altered the organism’s immunobiological reactivity, as manifested by decreases in the phagocytic index and phagocytic number.

2. Exposure to benzene at concentrations of 0.5, 0.2, and 0.05 mg/L for 3 months produced the typical picture of benzene intoxication in the experimental animals, manifested by leukopenia, thrombocytopenia, anemia, and reticulocytosis. At a benzene vapor concentration of 0.02 mg/L, mild leukocytosis was observed.

3. Changes in the phagocytic activity of leukocytes began before changes in the blood. Because of the high sensitivity of this reaction, its use as a test is proposed.

4. Recovery of leukocyte phagocytic activity was slow. At a concentration of 0.05 mg/L, phagocytic activity recovered only after 3 months.

5. The observed decrease in phagocytic activity at a benzene vapor concentration of 0.05 mg/L, the current maximum permissible concentration, and even at 0.02 mg/L indicates that these concentrations are not harmless to the organism.

These findings may provide grounds for reconsidering the existing maximum permissible concentration. This conclusion was also confirmed in subsequent work studying immunobiological reactivity in people working with benzene.

References

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Received October 11, 1957.