BIO-ASSAY OF T H E EFFECTS OF SMOKING ON DNA CONTENT OF HUMAN RESPIRATORY EPITHELIUM

DANIEL ROTH, MD, DONALD T. FREDRICKSON, MD, ABRAHAM OPPENHEIM, MD, AND MORRIS LONDON, PHD

A survey of alterations in the DNA content of human oropharyngeal epithelium related to smoking was carried out. The technique used had been developed for mass screening of the DNA content of populations of intact cells and was based on the binding of acriflavine by cells in proportion to their DNA content. Groups of nonsmokers, heavy smokers and cancer cases served to establish the normal and abnormal dye-binding ranges. Among 14 previously heavy smokers who abstained completely, there was a decline to the normal range in 6 months. The implications of this finding are discussed in connection with carcinogenesis and the application of the technique as a bio-assay in humans for evaluating cigarettes claimed to be safe is suggested.

URRENT INTEREST IN DEVELOPING A “SAFE” C cigarette makes it necessary to establish meaningful criteria for safety. At present the only criterion widely applied is the content of tar and nicotine in cigarette smoke. While experimental evidence supports their prob- able role in carcinogenesis, confirmatory proof is lacking and there remains a need for more direct means of measuring the capabil- ity of cigarettes to cause human cancer.

This report describes our procedures and observations in measuring changes in the DNA content of epithelial cells of the hu- man oral cavity related to cigarette smok- ing. The resultant data, coupled with the applicability of the procedure for large-scale surveys, offer the prospect of evaluating cig- arettes on the basis of their effect upon DNA content in the upper respiratory tract of man and thereby of assessing a biological event having fundamental significance with regard to carcinogenesis.

From the Department of Pathology, St. Barnahas Hospital for Chronic Diseases, New York, and the Division of Cancer Control and Research of the New York City Department of Health.

Supported by the Bureau of Adult Hygiene of the New York City Department of Health.

The authors express their appreciation to Dr. John Daly, Chairman of the Department of Otolaryngology, New York University, Bellevue Medical Center, for making clinical material available. Acknowledgment of the technical assistance of Mrs. Maria Manjon is also made. The statistical analysis was performed by Mr. Alex Tytun of the Division of Statistics, New York City Department of Health.

Address for reprints: Pathology Department, Uni- versity Hospital, 560 First Ave. New York, N.Y. 10016.

Rcccived for publication October 19, 1967.

METHODS AND MATERIALS

The analytical method and the harvest- ing of cells has been reported previously.l* Harvesting, in order to achieve maximal public acceptance and convenience, is self- performed and has been modified by includ- ing an initial phase of gargling with the saline solution before irrigating the anterior oral cavity. The procedure thus samples the oropharynx, fauces and oral cavity; a 30- second irrigation is sufficient to obtain ample numbers of cells in the majority of instances. No fixatives are used, samples being deliv- ered directly or mailed to the testing labora- tory. We have found that there is no signifi- cant deterioration of squamous cells at room temperature for u p to 4 days; indeed, in the absence of fixation, leukocytes, which are invariably present and can contribute a non- specific spurious increment of DNA to the analyzed sample, are autolyzed. Their solu- bilized DNA is then eliminated during the subsequent washing steps. The need for differential cell filtration, as described in our earlier publication, is therefore removed, with a resultant saving of time and expense.

After counting by hemacytometer suffi- cient volumes of cell suspension are drawn u p to provide the predetermined 50,000 cells to be analyzed. These are then washed twice in phosphate buffer (pH 6.0 to 6.2) in stand- ard centrifuge tubes, allowed to drain by in- version, then exposed to dilute acriflavine, as previously described. The cells bind dye in proportion to their DNA concen-

1132

BIO-ASSAY OF THE EFFECTS OF SMOKING Roth et al. 1133 No. 6

tration.18119 They are then spun down and saved for confirmatory Papanicolaou staining if indicated, while the supernatant dye is measured by fluorometry. This step may be expedited by use of one of the available auto- mated techniques of. photometry.17 The su- pernatant dye concentration is the negative function of the amount bound to the cells. The final result is recorded in picograms of dye bound per cell (10-12 Gm). Mathemati- cal calculations in the procedure are simpli- fied by using the formulae:

1. ml of initial suspension required for

No. of cells counted in 4 large hemacytometer squares

and

2. Picograms of dye bound per cell = % of dye adsorbed x 4.4 (Derived by divid- ing the amount of adsorbed dye by the number of cells, or: % dye adsorbed ~22x104 picograms dye

[original dye aliquot] 5x104 cells)

Human study groups were composed of:

1. 92 adult nonsmokers,

2. 74 heavy smokers (defined as smoking at least 1 % packs of cigarettes a day for the past 5 years).

3. 14 previously heavy smokers from group 2 who voluntarily stopped smok- ing completely for the entire 6-month period of the study.

4. 21 cases of proven squamous carci- noma of the oral cavity whose cells were

analysis = 20

* A comparison was made between the initial readings and the 6-month readings using Student’s t- test. The average difference for the 14 individuals is

- 2 A A = - = 5 . 4 - = 0.386. TZ 14

The standard error of ii is given by ZA2 3.84

8 2 = d- = 4- = 0.145 ~ ( T Z - 1) 14 X 13

The value ofLStudent’s t is ’ S x 0.145

t = - = - - 0‘386 - 2.66, with d.f. = 13.

This value of t is signscant at the 5% level. The average dye binding response was significantly lower after the 6 month abstention.

harvested in the manner described, as well as by gentle direct swabbing of the tumor. In a number of these cases the samples were handled by a double blind procedure; in all cases, the analyses were done by a technician who had no infor- mation other than their identification number.

The smokers and voluntary abstainers were recruited from the participants in a smoking withdrawal clinic conducted by one of us (DTF) under the auspices of the New York City Department of Health. These partici- pants, for the most part, represented the up- per middle class and were drawn from a cross-section of the metropolitan business and professional world. No medications were prescribed or recommended. No personal or medical history other than that relating to smoking was taken. Most of the individuals had smoked for 15 or more years. The non- smokers in the study were selected on the basis of a negative reply to the question “Do you smoke?” No other personal or medical history was elicited.

RESULTS

Table 1 presents the dye-binding data obtained in each abstainer over the 6-month course of the study. There is a significant de- cline in eight of the 14 cases. The 5 which showed no significant decline had been in the normal range from the start, as had the one showing a slight (insignificant) rise.”

TABLE 1 . Dye-binding Response in Abstainer Group*

Start of test Sixth month

3 2 2.0 - .~ 2.6 2.5 2.5 2.4 2 3 2.2 2.2 2.2 2.0 2.0 2 . 0 1.9 1.9

1.9 1.9 1.8 1.9 2.1 2.1 1.6 1.8 2.0 1.8 1.6 2.1 1.9

* Dye-binding values of each of the 14 individuals in the study group at start of test and a t 6 months. All values recorded in picograms (10-’2 Gm) dye per cell. The decline is greatest in those with the highest initial levels and diminishes as the initial levels cor- respond closer to the normal range.

1134 CANCER June 1968 VOl. 21

Fig. 1 presents the range of dye-binding over the 6-month period for the test group and over a 3-month period for 14 of the orig- inal smoker controls taken at random. Mean levels are depicted by the circles on ordi- nates. The ranges (950/, limits) for the 92 nonsmoker and 74 smoker controls are also shown. Included is the mean and the range of the group of 21 proven cases of squamous carcinoma of the oral cavity whose cells were harvested in the same manner as the study and control groups.

A reproducibility range of 0.1 picograms of dye bound per cell had been established for the analytical technique. Thus the mean decline of 0.4 picogram for the test group can be significant. The unchanged mean level for the smoker control group gives added confidence to these observations. Thus i t is apparent that the increased dye-binding of the oral epithelium of heavy smokers was reversed during a 6-month period of total abstinence.

DISCUSSION

The affinity of diaminoacridines for nucleic acids has led to a large literature covering a broad range of applications in clinical as well as in basic research into the structure of DNA,

the metabolism of polynucleotides and gene- directed intracellular phenomena.2. 41 5, ', &lo+

12-15, 21, 2% 24, 25 We have previously reported conditions for the specific binding of acri- flavine to cellular DNA in order to permit measurement of native DNA.Ig

When the binding of acriflavine is taken as an expression of the DNA content of popula- tions of intact oral cells it is surmised that one adverse effect of cigarette smoking is its ability, unexplained at present, to cause an increase in their DNA content. This phe- nomenon has already been demonstrated in connection with the lower respiratory tract11 and it is reasonable to assume that smoking af- fects the entire respiratory tree in a similar fashion. The increase in DNA could result from polyploidy, polyteny, increased cell turnover, prolongation of DNA synthesis and/or of mi- tosis. Depolymerization of native DNA or an increase in available binding sites on the DNA molecule could theoretically augment the dye- binding without an actual quantitative in- crease in DNA. Studies to clarify this point are currently in progress.

The greatest theoretical importance devel- oped by this study lies in the possibility that sustained high levels of cellular DNA, resulting from continued smoking, may be related to the ultimate development of squamous cancer of

cancers 'r T smokers -r 1

1 [ s i k e r s A 1

rna.1 rno.3

CONTROLS

1 rno.1 rno.3 rno.6

ABSTAIN E R 5

1 m o . 9

I I

FIG. 1. Dye-binding levels of control groups and study groups, showing the range and mean for each. The smoker control group of 14 is unchanged after a 3-month interval. The study group shows a decline. At the 6-month point all members of this group show values within the normal range, although the mean is still above that of the nonsmoker controls. This decline was maintained, as shown by the data obtained at the 9-month point from 11 respondents,

No. 6 BIO-ASSAY OF THE EFFECTS

the oral mucosa. In a series of 21 proven cases of oral cancer the mean and median dye-bind- ing values for the cells harvested by irrigation of the total mucosal surface was 3.2 picograms per cell. When cells were obtained by direct scraping of the surfaces of the actual tumors the mean value was 3.3 picograms and the median 3. Thus, the fundamental quality of the “field alteration” which is generally believed to oc- cur and to predispose the oral cavity to mul- tiple and recurrent carcinogenesiszo may be this diffuse increase in the DNA content of the whole cell population.

We have found a significant but temporary increase in dye-binding of pharyngeal epithel- ium during acute viral pharyngotonsillitis, subsidence to normal levels requiring about 3 weeks. It is conceivable that continued smok- ing, in some unknown manner, may either alter DNA metabolism directly or may cause rela- tively permanent retention of exogenous DNA incorporated into the oral epithelium from a wide variety of sources, including viral.

The return towards normal upon termina- tion of smoking suggests a central and control- ling role for smoking. This reversibility of the increase in DNA content of oral epithelium upon cessation of smoking appears to have its experimental parallel in such studies as those of Tipton and Crocker23 who demonstrated a return to normal of the metaplasia induced in the bronchial epithelium of dogs by conden- sates of cigarette smoke after discontinuation of this treatment. The gradual nature of the decline may reflect the several cell generations required for deletion of the abnormal effects from the basal epithelium (the most probable target of the injury) together with the gradual progression of the injured cells toward the sur- face of the squamous epithelium before their final exfoliation and harvesting.

Perhaps from these studies there may be de- veloped a method for monitoring the heavy smoker by “titrating” the number of cigarettes he can consume without raising the DNA con- tent of his oral mucosa to some presently un- defined danger level. Below this level he may be kept relatively secure from future carcino-

OF SMOKING Roth et al. 1135

genesis. At present a sharp “threshold” level of 2.2 picograms of dye per cell seems to be emerg- ing, representing the upper limit for normal nonsmokers and the lower limit for cases of cancer. Final establishment of a quantitative discriminant awaits a larger statistical sam- pling and the long-range follow-up of cases with persistently high levels of dye-binding. It should be pointed out that transient elevations can occur during acute viral pharyngotonsillitis and in any condition which may increase the DNA content of cell populations, e.g., rapid regeneration, trisomy as in Down’s syndrome, irradiation injury, etc.

Within these limitations on interpretation this approach is suggested as a means for asses- sing devices intended to make cigarettes “safe.’ Serving as a direct bio-assay in humans sub- jected to normal smoking conditions, it would meet the objections of those who will not equate results obtained in the experimental laboratory with the living human state.l? 6, 16, 23 Since squamous carcinoma of the oral cav- ity is a “smoker’s” cancer as is bronchogenic cancer, it would be reasonable to interpret data obtained from the oral mucosa as pos- sessing significance for the lower respiratory tract as well.

Certainly one cannot with finality judge a cigarette’s carcinogenic potential merely by measuring changes in DNA content of respira- tory tract epithelium. However, even in the face of our incomplete comprehension of the etiology and pathogenesis of cancer, a demon- strated decline toward the nonsmoker level of epithelial cell DNA which might be brought about by a new cigarette filter or other device would be welcome. If this were concomitantly associated with a reduced tar and nicotine con- tent, one would be on firmer ground in claim- ing safety for his product. Such a study is currently in preparation.

ADDENDUM

Data provided at the 9-month point by I 1 respond- ents of the original group of 14 are included in Fig. 1. They demonstrate no change from the 6-month levels, ranging between 1.6 and 2.1 picograms, with a mean of 1.9.

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