Hematologic and clinical chemistry effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals.

Chlorodibenzo-p-dioxins, especially 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), are among the most toxic compounds known. These compounds are found as contaminants of technical chlorophenols and their derivatives. A variety of pathologic condition have been associated with the ingestion of products containing chlorodibenzo-p-dioxins. Toxic fat, the cause of chick edema disease (1, 2) and a variety of pathologic manifestations in monkeys (3), has been found to contain chlor3dibenzo-p-dioxins, among which is TCDD (4, 5). TCDD has been implicated in a variety of other toxicoses, including outbreaks of chloracne in chemical workers (6), hepatonecrosis in rabbits (6), and hepatonecrosis and thymic atrophy in rats (7). However, only in the studies in which monkeys were fed toxic fat (3) and in which rats were given TCDD orally (7, 8) were substantial clinical pathologic analyses performed. Toxic fat caused in the monkeys, among other lesions, anemia, leukopenia and hypoproteinemia. The hypoproteinemia was primarily due to decreased serum albumin concentration. The blood urea nitrogen


Introduction
Chlorodibenzo-p-dioxins, especially 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), are among the most toxic compounds known. These compounds are found as contaminants of technical chlorophenols and their derivatives. A variety of pathologic condition have been associated with the injestion of products containing chlorodibenzo-p-dioxins.
Toxic fat, the cause of chick edema disease (1, 2) and a variety of pathologic manifestations in monkeys (3), has been found to contain chlorodibenzo-p-dioxins, among which is TCDD (4,5). TCDD has been implicated in a variety of other toxicoses, including outbreaks of chloracne in chemical workers (6), hepatonecrosis in rabbits (6), and hepatonecrosis and thymic atrophy in rats (7). However, only in the studies in which monkeys were fed toxic fat (3) and in which rats were given TCDD orally (7,8) were substantial clinical pathologic analyses performed. Toxic fat caused in the monkeys, among other lesions, anemia, leukopenia and hypoproteinemia. The hypoproteinemia was primarily due to decreased serum albumin concentration. The blood urea nitrogen (BUN), serum bilirubin, cholesterol, sodium and potassium concentrations, and the prothrombin times were not altered (3).
Rats given 10 mg TCDD/kg body weight had, among the other lesions, increased activities of serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvate transaminase (SGPT), lactic dehydrogenase (LDH), and hydroxybuturate dehydrogenase as well as decreased arylesterase and cholinesterase activities. In addition, the rats had decreased serum glucose, sodium, and protein concentrations, and increased serum urea, lipid and bilirubin concentrations, hemoconcentration, and neutrophilia. Serum cholesterol, potassium and chloride concentrations and aldolase and alkaline phosphatase (AP) activities were not altered (7,8).
Because the clinical pathologic and pathologic changes found in these and other studies suggest that the most profound effect of chlorodibenzo-p-dioxins are on the liver and various hematopoietic organs, we decided to determine the sequential clinical pathologic changes which might occur in rats given different doses of TCDD over a period of time. These methods might also determine which functions are the most sensitive to TCDD. In addition, hematologic .studies were conducted as an adjuvant to studies of immunologic effects of TCDD in guinea pigs (9) and in mice in which the effects of TCDD on o6genesis are being determined (J. McLachlan, personal communication).

Animals
Female CD rats weighing 150-175 g were given, orally, 0.1, 1.0, or 10.0 Ixg TCDD/kg daily for 30 days. Blood for the clinical pathologic studies was obtained from the heart 3, 6, 10, 13, 17, 24, and 31 days after the TCDD dosing commenced. Blood was obtained from the retroorbital sinus for hematologic studies 1, 3, and 5 weeks after 8-week-old female CD-1 mice were given oral doses of TCDD of 1.0, 10.0, or 50 ,g/kg.
Female Hartley strain guinea pigs weighing about 250 g received oral weekly doses of 0.008, 0.04, 0.2, or 1.0 ug TCDD/kg for 8 weeks. The 1.0 jug TCDD/kg group became moribund between 3 and 5 weeks. Half the animals were injected with tetanus toxoid (group A) and half with killed Mycobacterium tuberculosis (group B) in order to determine the effects of TCDD on humoral and cell-mediated immune responses. The details of these experimental procedures can be found elsewhere in this issue (9). The highest dose guinea pigs when moribund and the other animals at 8 weeks were bled from the heart for hematologic analysis.

Procedures
Routine methods were used for hematologic determinations. Platelet counts were determined in counting chambers (Unopette, Becton, Dickinson and Company, Rutherford, N. J.), fibrinogen by a heatdenaturation method (10); urea nitrogen was determined by a urease method (Boehringer-Mannhein Co., New York, N. Y.), cholesterol by a ferric chloride method (Hyland, Costa Mesa, California), serum proteins by the biuret technioue (11), glucose by an o-toluidine method (Hyland, Costa Mesa, California), creatiniine by an alkaline picrate method (12), bilirubin by a sulfanilic acid-caffeine-sodium benzoate method (Boehringer-Mannheim Co., New York, N. Y.), sodium and potassium by flame photometry, and chloride by coulometric titration. The serum enzymes, alkaline phosphatase (AP), lactic dehydrogenase (LDH), glutamic-oxaloacetic transaminase (SGOT), and glutamic pyruvate transaminase (SGPT) were determined by kinetic methods by use of commercially available kits (Sigma Chemical Co., St. Louis, Missouri) in a recording spectrophotometer. Analysis of Data Statistical analysis for differences between groups was done by Dunnett's test (13). Determination of a dose response was done by Jonckheere's method (14). The 5% level was selected as the value where variables would be considered significantly different.

Rats
Enzymic alterations in the TCDD-treated rats consisted of increased SGPT activity in the high-dose (10.0/ug TCDD/kg-day) rats at days 17, 24, and 31 ( Fig. 1), and increased SGOT activity at days 13, 17, 24, and 31 in the high-dose rats as well as days 13   Environmental Health Perspectives elevated only on day 24 in the high-dose rats, and AP was not altered at any time.
Serum cholesterol was increased on days 10, 17, 24, and 31 in the high-dose rats and day 24 in the middle-dose animals. Significant dose-response elevations for cholesterol concentrations were also found on days 17 and 24 (Fig. 3). Blood glucose concentration decreased with time in all the treated groups. At day 10, glucose concentrations in all treated animals were significantly decreased and on days 24 and 31 in the high and middle dose animals it was decreased. Significant dose-response blood glucose decreases were found at days 10, 17, and 24 (Fig. 4). Total serum protein fluctuated with the treatment. On days 24 and 31, the high-dose rats had decreased protein concentration while at day 31 it was increased in the middle-dose rats (Fig. 5). In addition, serum bilirubin concentrations were increased in the high-dose animals on days 17, 24, and 31 (Table 1). No significant changes were found for serum creatinine, sodium, potassium, chloride, fibrinogen, and BUN concentrations.  Hematologic changes were confined to hemoconcentration, as previously noted in rats (7), in the high-dose animals on days 17 and 24 (Figs. [6][7][8], and to striking thrombocytopenia in all the groups. After only 3 days treatment, the high and middle-dose animals had depressed platelet counts which remained depressed throughout the study. In the low-dose rats (0.1 jug TCDD/kg-day), platelets were decreased "E 10  significantly only on day 17. Significant dose response decreases in platelets occurred throughout the study (Fig. 9). No significant changes occurred in leukocyte counts or differentials (in particular, lymphocytes) in these rats.

Mice
Mice given a single oral dose of 1.0, 10.0, or 50.0 ug TCDD/kg had significantly decreased leukocyte (Table 2) and lymphocyte counts (Table 3) after 1 week. After 3 weeks, none of the treatment groups had significant differences from control mice; however, a significant dose-response lymphocyte depression remained. The leukocyte depression was on the borderline of being a Environmental Health Perspectives significant dose-response. After 5 weeks, no TCDD effects were detected except that the mice treated with 10 jug TCDD/kg had significantly elevated erythrocyte counts ( Table 4). This difference is probably due to a lack of variability in the parameter rather than a real elevation and likely does not reflect an effect of TCDD.

Guinea Pigs
The TCDD-treated guinea pigs in the tetanus toxoid-injected group (group A) (9) had consistently lower leukocyte counts than the controls, but only for the middledose animals (0.04 jug TCDD/kg) was this decrease significant ( Table 5). The lymphocyte counts were significantly decreased in all the TCDD-treated animals in this group. A significant dose response occurred for the leukocyte and lymphocyte decreases also, but these effects simply reflect the depression observed at all three TCDD treatment levels.   For Group B (Mycobacterium tuberculosis tuberculin-treated) (9) only the highest dose (0.2 ,g TCDD/kg) caused significantly decreased leukocyte counts (Table  6). Neutrophil counts were also reduced in these guinea pigs, but there was no treatment versus control differences in lymphocyte counts. Significant dose-related decreases were found for leukocytes, neutrophils, and lymphocytes. In addition, the guinea pigs treated with 0.2 4g TCDD/kg also had lower platelet counts than the controls (P< 0.01).
The leukocyte and lymphocyte counts of the group A and group B control guinea pigs did not differ significantly (Table 7). However, the group B controls did have higher neutrophil counts than the group B controls (P< 0.05). When the group A control guinea pigs were compared with the moribund 1.0 /g TCDD/kg guinea pigs, decreased lymphocyte and increased neutro-Environmental Health Perspectives phil counts were found in the treated animals. However, this comparison may not be valid because the TCDD-treated guinea pigs were 3 to 5 weeks younger than the group A controls and were in a moribund state in addition to not having been injected with tetanus toxoid. Hemoconcentration was also present in these dying animals, there being an average erythrocyte count of 5.1 x 106 per mm3 and a hematocrit of 48.9%o. Platelet counts were decreased as well.

Discussion
The clinical pathologic findings of this and other studies (3,7,8) suggest that the major sites of the toxic action of TCDD and the chlorodibenzo-p-dioxins of toxic fat are the hematopoietic system and the liver. Mice and guinea pigs given TCDD and monkeys fed toxic fat (3) were leukopenic, which in mice and guinea pigs is characterized by lymphopenia. In mice which received a single dose of TCDD the lymphopenia was reversed 5 weeks after TCDD exposure. Perhaps the lymphopenia seen in mice and guinea pigs is related to the decreased cell-mediated immune response observed in these two species after TCDD exposure (9). It is interesting that lymphopenia and cell-mediated immunosuppression were not found in rats treated with TCDD.
Rats and guinea pigs were thrombocytopenic. The cause of this was not determined. Examination of rat bone marrow in one experiment did not reveal any differences in numbers or morphology of megakaryocytes following TCDD treatment (17), but in another experiment in rats, spleen and bone marrow megakaryocytes were degen-erated and appeared to be reduced in numbers (15). Therefore, further work is indicated to determine, perhaps by radioisotope tagging, whether the lowered platelet counts are due to decreased production or increased distruction of platelets. Nevertheless, thrombocytopenia or defects in the clotting mechanism (17) might have played a role in the production of hemorrhages that were occasionally seen in rats that died (15).
Anemia was not found in these studies, but it was seen in a previous study in monkeys fed toxic fat (3). That anemia was accompanied by atrophic bone marrow and normal serum bilirubin suggesting that the anemia was aplastic or depression type rather than hemolytic anemia.
We observed only terminal hemoconcentration in rats and guinea pigs, similar to that previously reported in rats given TCDD (7). This is probably an effect of terminal shock and dehydration rather than increased erythrocyte production. Dehydration is further evidenced by the increased serum protein concentrations found in the middle-dose rats. However, hypoproteinemia was observed in the high-dose rats probably as a result of liver damage, and may have contributed further to the hemoconcentration by decreasing the colloidal osmotic pressure of the blood and allowing fluid to accumulate in the tissues.
Liver damage in rats was evidenced by increased SGOT and SGPT activity. The increased SGOT activity might also be due to, in part, myocardial necrosis (15). Hyperbilirubinemia, hypercholesterolemia, and hypoproteinemia (in the high-dose rats) are probably other effects of the liver pathology. Since increased cholesterol concentrations have previously been seen in rats (18) and rabbits (19) which have sustained hepatocellular damage from polychlorinated biphenyls, perhaps metabolism of cholesterol is altered in liver damage caused by these chlorinated compounds. Hypoglycemia in the TCDD-treated rats might have been due to decreased food consumption (16), but there might also be an effect on the gluconeogenic ability of the damaged liver. Although clinical chemical parameters to assess liver damage were not determined in guinea pigs and mice, histopathologic evidence suggests that hepatocellular damage was minimal in these species (15).
It appears that hepatocellular necrosis is the main toxic action of TCDD in rats but with effects on platelets being important. The clinical pathologic changes are consistent with this hypothesis. Ultimately, hemoconcentration due to shock and dehydration occurs as a terminal event in rats as well as guinea pigs.
The lymphopenia observed in mice and guinea pigs are consistent with the immunosuppressive effects of TCDD in these species (9). Pathologic studies suggest that liver damage does not play a part in the death of guinea pigs given high doses of TCDD (15). However, in guinea pigs, atrophy of the adrenal zona glomerulosa (9,15) suggests that electrolyte imbalance might occur. Perhaps a study of serum electrolyte and aldosterone concentrations in guinea pigs given lethal doses of TCDD is warranted.