Respiratory Activity and Nucleic Acid Content of Some Tissues from Guinea Pigs Sensitized to 2,4-Dinitrochlorobenzene1

RESPIRATORY ACTIVITY AND NUCLEIC ACID CONTENT OF SOME TISSUES FROM GUINEA PIGS SENSITIZED TO 2, 4DINITROCHLOROBENZENE* LEON L. GERSHBEIN, PH.D., BOGUSLAW K. KROTOSZYNSKI, PH.D., MATTHEW J. BRIJNNER, MI). AND A. J. T)OMNAS, M.S.

As an approach to the study of the mechanism of eczematous sensitization, a Warburg study was undertaken of the respiratory activity of skin and reticuloendothelial tissues of guinea pigs sensitized with 2, 4-dinitrochlorobenzene (DNCB). It was thought that tissues of such sensitized animals might exhibit changes in Q2 (microliters of oxygen taken up by 1 mg. dry tissue per hour) on introducing the antigen into the system. However, such behavior was not noted. In addition, no significant changes were found in nucleic acid levels of skin (DNA) and spleen (DNA and RNA) from sensitized animals as compared with the control values. METHODS

Eczematous sensitization was engendered in albino guinea pigs of either sex nd ranging 300—450 g. in weight by application of a solution of 10 % DNCB in acetone to the clipped skin of the lower abdomen. The DNCB (Eastman Kodak Company) was recrystallized prior to use. The antigen was applied on each of five alternate days. Primary irritant reactions were observed but cleared by the 18th day after the last application, when challenging was carried out. For the latter, concentrations of 0.01, 0.1 and 1.0% DNCB in acetone were administered to the lower right, upper right and upper left dorsal sites, respectively. The areas were rated on the 24th and 48th hours and only those animals showing marked

inflammatory reactions to both the 0.1 % and 1.0% dosages were classed as sensitized and employed in subsequent work; doubtfuls or negatives were excluded. Generally, reactions ranging from 1 + to 4+ and 3+ to 4+ were evident with the 0.1 % and 1.0 % solutions, respectively; at the 0.01 % level, ratings extended from negative or doubtful up to 1+. For these experiments, all guinea pigs were sacrificed by decapitation and the requisite tissues (skin from the unchallenged or lower left dorsal site, spleen, inguinal lymph nodes and bone marrow) removed and placed in ice-chilled Krebs-Ringer-phosphate medium of pH 7.4 (1). Skin specimens from the various sites were reserved for histological examination. All tissues were trimmed of fat

prior to slicing. For bone marrow, the large bones were broken and the tissue removed and used without further preparation. Skin was cut into fine strips by * From the Biochemistry Research Laboratory, Department of Biology, Illinois Institute of Technology and Department of Dermatology, Northwestern University School of Medicine, Chicago, Illinois. This study was aided by a grant from the Toni Division of the Gillette Company, Chi-

ago, Illinois. Received for publication February 24, 1956. 73

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a specially designed press and then reduced to small fragments with scissors. Spleen and lymph nodes were sliced by hand with razor blades according to conventional methods. The weighed tissue slices were introduced into the Warburg flasks containing 1.8 ml. Krebs-Ringer-phosphate in the main chamber, 0.2 ml. 20% NaOH in the center well and 1 ml. of a fine suspension of 0.1 mg. DNCB in buffer in the side-arm. Pure oxygen was used in the gassing of the system. Except for lymph nodes and bone marrow, the tissues were not pooled. In each case, the Qo was determined before and after introduction of the antigen into the main chamber. Many trials were carried out to ascertain the optimum concentration of DNCB and the most suitable medium for the Warburg studies. Among others, these included saturated solutions in water, saline or buffers, suspensions produced by mulling the agent with gum tragacanth in water and propylene glycol or alcohol mixtures. The composition selected (0.1 mg. DNCB/ml. Krebs-Ringer-phosphate) proved most suitable, causing only occasional small decreases in Qo2 of normal tissues. Higher levels of DNCB were quite toxic as evidenced by drastic diminution of Qo2 values.

The nucleic acid determinations were based on the phosphorus content (2). The dry tissue powders, prepared according to the method of Schmidt and Thannhauser (3), were employed for the analysis of skin DNA and splenic total nucleic acid and DNA; RNA for spleen was obtained by difference. As the dry skin powders were not sufficiently homogeneous for the relatively mild extraction

procedure required for total nucleic acid evaluation, only the DNA content which involves tissue hydrolysis was determined. RESULTS

Nucleic acid levels in terms of mg. P/100 g. fresh skin or spleen and the average Qoz values for spleen, skin (from unchallenged site), lymph nodes and

bone marrow before and after the introduction of DNCB together with the Fisher t values are given in Tables 1 and 2, respectively. From the present data, it will be noted that no significant alteration in nucleic

acid content of spleen and skin accompanied sensitization (Table 1). The Q for spleen, skin, lymph nodes and bone marrow did not differ markedly from those of the respective controls before the "tipping in" of DNCB (P > 0.05 for each comparison except for bone marrow where the number of runs was insufficient for an adequate statistical calculation). This was also the case in the com-

parison of corresponding values obtained after the introduction of the agent. Although decreases in Qo2 were definite with spleen slices, these were of the same order of magnitude (12—14%) in both sensitized and control tissues. The diminution in Qo2, therefore, stems from a toxic action of DNCB and is not dependent on sensitization.

It is of interest that the respiratory activity of skin removed from the upper right dorsal area (challenged with 0.1 % DNCB-acetone solution), ranged signifi-

cantly higher than that from the unchallenged site or from a similar one in the control animals. The Qo2 S.E. values before and after introduction of DNCB

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RESPIRATORY ACTIVITY AND NUCLEIC ACIDS OF GUINEA PIG TISSUES

TABLE 1 Nucleic acid content of control and DNCB-sensitized guinea pig skin and spleen DNA

RNA

mg. P/too g. fresh tissue SE.

mg P/leO g. fresh tissue S.E.*

No. of

Treatment

Animals

Skin

Controls...... Sensitized

17 15

0.56

6.31 7.31

0.81

0.86

Spleen Controls

21

45.50

Sensitized

21

35.92

* S.E., standard t

P

4.85 3.47

1.61

49.29 49.78

1.90 4.15

0.01

error.

> 0.05.

TABLE 2 Q02

value for control and sensitized guinea pig skin, spleen, ingninal lymph nodes and bone marrow Qo,

Treatment

S.E.

Of

nlma

Before tipping

Afte1 iin in

Spleen Controls Sensitized

24 24

4.67 4.74

0.17 0.15

4.12

0.16

4.09

0.16

0.36 0.43

0.05

0.40 0.48

0.04 0.05

0.61

0.16 1.17 0.14 1.20

0.16

0.23 0.08

4.22 4.02

0.73 0.34

2.39t

3.02

Skin Controls Sensitized (unchallenged site)....

24 24

0.04

0.62

Lymph nodes

11

Controls Sensitized

12*

1.12 1.19

0.11

Bone marrow

3

Controls Sensitized

3*

4.67 4.67

0.61 0.16

* Microliters of oxygen absorbed per milligram of dry tissue per hour. t Comparison of average Qo2 before tipping with the corresponding value after introduc. tion of I)NCB. P < 0.05. §

Pooled

tissue from at least 3 animals.

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were 0.62 0.08 and 0.61 0.10 (9 animals), respectively, while the corresponding control figures for the same number of guinea pigs amounted to 0.42 0.03 and 0.39 0.04 in the order given. Again, DNCB was found to have no signifi-

cant influence on the resniratory activity. Although it was presumed that the elevated Qo, for skin from the challenged site might be due in large measure to lymphocytic infiltration of the "damaged" area, this could not be unequivocally substantiated by microscopic examination of such sections. The above behavior may also be attributed to an increased rate of epidermal proliferation at the site of the dermatitis. More useful information toward mechanism elucidation might be forthcoming

from similar work but employing conjugates of DNCB with skin and other proteins, instead of the simple nitro-compound. As it is likely that some metabolic activities may be affected by the sensitizer without concomitant profound changes in oxygen uptake, a study of specific intracellular enzymatic systems may be even more revealing. An investigation of the effect of DNCB-protein conjugates and other antigens of lower toxicity on the respiratory activity and enzyme levels of various tissues is in progress. SUMMARY

1. In a Warburg study, lymph nodes, spleen, skin (from unchallenged site) and bone marrow from guinea pigs sensitized to 2, 4-dinitrochlorobenzene displayed no noteworthy differences in respiratory activity (Q02) over the controls, either before or after the introduction of sensitizing agent. 2. The mean Qo, of skin from sites challenged with a 0.1 % solution of 2, 4-dinitrochlorobenzene in acetone, ranged significantly higher than the value for the

corresponding controls and is probably unrelated to the sensitization phenomenon. 3. The nucleic acid levels of skin (DNA) or spleen (DNA and RNA) remained unaltered on sensitization. REFERENCES 1. UMBREIT, W. W., Burtxis, R. H. AND STAUFFER, J. F.: Manometric Techniques and Tissue Metabolism. Minneapolis, Burgess Publishing Company, 1949. 2. LOGAN, J. E., MARINEL, W. A. AND RossITER, R. J.: Estimation of nucleic acids in tissue

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