The Laryngoscope
Lippincott-Raven Publishers, Philadelphia
0 1997 The American Larvngolorncal.
Rhinological and Otological soci&y, Inc.
Genetic Susceptibility to Head and Neck
Cancer: Interaction Between Nutrition and
Mutagen Sensitivity
Stimson P. Schantz, MD; Zuo-Feng Zhang, MD, PhD; Margaret S. Spitz, MD, MPH;
Ming Sun, MS; Tao C. Hsu, PhD
The development of head and neck cancer may
depend not only on exposure to environmental carcinogens but also on a genetically based susceptibility to carcinogen-induced damage. This thesis
presents a case-control study that demonstrates the
significance of mutagen sensitivity, a measure of an
individual’s intrinsic DNA repair capacity against
free radical damage, as a risk factor for the disease.
As part of the case-control analysis, 167 previously
untreated patients and 177 age- and sex-matched
healthy controls were assessed for various lifestyle
factors including tobacco and alcohol habits, occupational exposures, and diet. Mutagen sensitivity
expressed by each individual was determined
by quantifying bleomycin-induced chromosomal
breaks within peripheral blood lymphocytes in
vitro. Consistent with our initial observations and
those of others, mutagen hypersensitivity was
strongly associated with increased risk of head and
neck cancer (odds ratio, 4.95;95% confidence interval, 2.67 to 9.17) after adjusting for age, sex, and
race. Low intake of vitamins C and E was also associated with an increased risk of disease and was interactive with mutagen sensitivity in risk estimates. Individuals with both a low intake of
various antioxidants and increased chromosomal
sensitivity to oxidant-induced DNA damage were at
greatest risk. This study supports the concept that
the risk of head and neck cancer is determined by a
balance of factors that either enhance or protect
From the Head and Neck Surgery Service (S.P.S.) and the Epidemiology and Biostatistics Service (Z-F.Z.), Memorial Sloan-Kettering Cancer
Center, New York, New York and the Departments of Epidemiology
(M.S.S.) and Cell Biology (T.C.H.),
M.D. Anderson Cancer Center, Houston,
Texas.
Presented a s a Candidate’s Thesis to the American Laryngological,
Rhinological and Otological Society, Inc.
This study was supported by grant no. R01 CA51845 from the National Cancer Institute.
Send Reprint Requests to Stimson P. Schantz, MD, Memorial SloanKettering Cancer Center, 1275 York Avenue New York, NY 10021, U.S.A.
Editor’s Note: This manuscript received the Harris P. Mosher Award.
Laryngoscope 107: June 1997
against free radical oxygen damage, including innate capacities for DNA repair.
Laryngoscope, 1OR765-781,1997
INTRODUCTION
Head and neck carcinogenesis may depend not
only on environmental exposures but also on an increased susceptibility to those exposures-an interaction between’both exogenous carcinogens and endogenous host influences. External factors include
levels of carcinogen exposure and other potential coetiologic agents, such as alcohol and the human papilloma virus.1-3 External factors that serve to diminish cancer risk, such as various nutritional
substances, may also be involved.4 The host factor
that forms the principal focus of this report is the individual’s innate capacity for DNA repair.5
In 1983, Hsu6 postulated that chromosomal
fragility, a measure of DNA repair capacity, was not
an all-or-none phenomenon. Rather, a gradient exists within the general population. Furthermore,
fragility would not be expressed spontaneously, but
hidden. Its existence would become apparent only
after clastogenic exposures. Hsu postulated that the
individuals who expressed the greatest degree of
mutagen-induced chromosome fragility would be at
greatest risk for cancer development. Such a process
would also account for the site of disease development. Only tissues exposed to mutagens would be at
risk. Thus the concept of susceptibility to mutageninduced chromosomal breakage would account for
diseases such as colon cancer, lung cancer, and head
and neck cancer. It would not explain other neoplastic processes in which environmental exposures are
not a principal feature, such as breast cancer, sarcomas, and central nervous system tumors.
To establish the relevance of chromosome
fragility (i.e., mutagen sensitivity) as a risk factor
Schantz et al.: Genetic Susceptibility to Cancer
765
for disease development, Hsu et al.7 developed the
bleomycin-sensitivity assay. Bleomycin had long
been known as a clastogenic agent. The mechanism
of its action had generally been considered to be radiomimetic (i.e., through the generation of free oxygen radicals), a fact relevant to tobacco carcinogenesis because numerous compounds within tobacco
condensate may generate free oxygen radicals capable of producing DNA double-strand breaks and
subsequent mutations.8-10 The assay they developed
(which forms the basis of this report) involves the
use of peripheral blood lymphocytes. In brief, lymphocytes are obtained from donor subjects and cultured for 5 days in the presence of the mitogen phytohemagglutinin. During the last 5 hours of the
culture, cells are exposed to the clastogen bleomycin, and chromosomal breaks are generated. To
quantitate the breaks, cells are arrested in mitosis
using Colcemid. The number of chromosomal breaks
per cell are then quantitated following analysis of 50
cellular metaphases. The mechanisms that account
for differences in chromosomal sensitivity to bleomycin among individuals are undoubtedly multiple,
including susceptibility to initial damage as well as
repair. 11J2
The first study to evaluate mutagen sensitivity
as a potential risk factor in head and neck carcinogenesis involved the assessment of 46 patients and
39 healthy controls.13 A statistically significant increase in bleomycin-induced breaks was noted in
the head and neck cancer patients. Seventy-two percent of the patients were noted to be hypersensitive
to bleomycin as compared with 28% of the controls.
The greatest differences in sensitivity to bleomycin
were observed when comparing individuals with
greater than 40-pack-per-year cigarette exposure
who remained disease free versus the head and neck
cancer patients with similar levels of exposures.
Similar findings regarding the resistance of elderly
individuals without a history of tobacco-induced disease had been previously reported by Hsu14 and Hsu
et al.15 The difference between a smoker without
cancer and one with head and neck cancer is that
the latter individual is abnormally sensitive to mutagens. Several reports followed that investigated
mutagen sensitivity expressed by head and neck
cancer patients.16-20 In each case similar findings
were noted. Head and neck cancer patients expressed increased sensitivity to mutagens. Furthermore, relevant to the conduct of the present casecontrol study, an interaction between tobacco
exposure, mutagen sensitivity, and head and neck
cancer could be identified. Individuals who both
smoked and were mutagen sensitive were at greatest risk.
Further evidence supporting the biologic and
clinical significance of mutagen sensitivity was provided by a prospective longitudinal follow-up study of
assessed head and neck cancer patients in which 84
Laryngoscope 107: June 1997
766
patients were evaluated prior to the treatment of
their index disease.21A wide range of mutagen-sensitivity values were noted, with patients expressing
both resistance and sensitivity to mutagen-induced
damage. It was postulated in this study that the risk
of second primary malignancies would be greatest in
the most sensitive individuals. The inherent inability
to repair DNA damage would increase the carcinogenic impact of tobacco exposure. The probability that
neoplastic transformation would involve multiple
cells would be increased. In support of this concept it
was noted that head and neck cancer patients who
were hypersensitive to mutagens expressed a 4.4-fold
higher risk for multiple cancers than more resistant
individuals in this prospective analysis.21 Subsequent
studies by Cloos et al.19 and Spitz et a1.22 have provided supportive evidence for this association.
RATIONALE FOR PRESENT CASECONTROL ANALYSIS
The purpose of the present study was to establish through a standard case-control design the significance of mutagen sensitivity as an independent
risk factor for disease. It was to support original observations cited above, as well as t o extend those observations by looking for interactions with other
lifestyle factors that may account for disease. A major focus of this presented case-control study is the
interaction of diet, mutagen sensitivity, and head
and neck disease. Numerous studies have indicated
the role of diet in either promoting or preventing
disease progression. The majority of these studies
have pointed to the protective effect of fruits and
vegetables (reviewed in Schottenfeldl and Wind). It
is postulated that this latter beneficial influence is
exerted through its antioxidant affect (i.e., the provision of free radical oxygen scavengers). High levels of such scavengers, including various vitamins
such as vitamins C and E, would prevent electrophilic genotoxic-induced damage.
The in uitro effect of dietary free radical scavengers on bleomycin-induced chromosomal damage
had been previously rep0rted.23~24In these latter
studies, lymphocytes from head and neck cancer patients were cultured in uitro and exposed to bleomycin. Varying concentrations of free radical scavengers, including vitamins A, C, and E, were
individually tested for their ability to block chromosomal breakage. In each case, increasing levels of
the respective scavengers inhibited bleomycin-induced damage. We hypothesized in this case-control
analysis that a similar process would occur i n uiuo.
Namely, fkee radical oxygen damage represents a
basis for cancer initiation. The overall probability of
developing disease would reflect a balance between
environmental exposure, nutritional habits that
may protect against that exposure, and an inherited
susceptibility to DNA damage. The significance of
such an epidemiologic study are twofold: to provide
Schantz et al.: Genetic Susceptibilityto Cancer
insight into further scientific investigations which
provide a mechanistic basis for head and neck cancer development and to provide a basis for targeting
prevention efforts to those at greatest risk.
MATERIALS AND METHODS
Cases
All patients entered into this study were diagnosed
with histologically confirmed squamous cell carcinoma
of the upper aerodigestive tract and were seen a t
Memorial Sloan-Kettering Cancer Center. Sites of disease were classified by the American Joint Committee
on Cancer criteria and consisted of oral cavity, pharynx, and larynx. All patients had been previously untreated. A total of 167 patients were identified between
the years 1992 and 1994.
Controls
One hundred seventy-seven age- (within 5 years) and
sex-matched controls were identified for this study. Controls were without previous history of cancer and were
identified within the Blood Bank Center of Memorial
Sloan-Kettering Cancer Center.
Data Collection
Each patient and control was asked to complete a
risk factor questionnaire. The questionnaire identified the
following variables: 1. demographic information: gender,
race, birth year, place of birth, religion, and education; 2.
dietary factors: ingestion of fat, vegetables, fruits, and
supplements such as vitamins C, E, and A; 3. occupational
and environmental exposures; 4. family history of cancer;
5. sexual behavior; 6. personal habits: cigarette smoking,
years of smoking, age a t initiation of smoking, alcohol consumption, types of alcohol, frequency and quantities of alcohol consumption, years of alcohol ingestion; and 7. medical and oral hygienic history. The validation of this
self-administered, comprehensive cancer risk factor questionnaire has been previously reported.25
Mutagen-Sensitivity Assay
The assay used in this study has been described in detail previously.15 A peripheral blood sample was collected
from each donor in a heparinized tube prior to the initiation
of lymphocyte culture. The standard lymphocyte culture
procedure used RPMI-1640 medium, supplemented with
15% fetal calf serum and phytohemagglutinin in a ratio of
blood to medium of 1:9.At 67 hours of incubation, one set of
cultures is treated with bleomycin (0.03 U/mL) for 5 hours.
Colcemid (0.04 mg/mL) is added in the last hour to induce
mitotic arrest prior to harvesting. Conventional cell harvesting procedure follows: the cells are treated with hypotonic KC1 (0.06M KC1) solution for 15 to 20 minutes, fixed,
washed with a freshly prepared mixture of methanol and
acetic acid (3:l) and air dried on wet slides. The slides are
stained with Giemsa solution without banding.
Fifty well-spread metaphases are examined from
coded slides. Chromatid aberrations are recorded as frank
chromatid breaks or exchanges. Bleomycin tends to induce
few chromatid exchanges (which if present, are considered
as two breaks). Chromatid gaps or attenuated regions are
Laryngoscope 107:June 1997
disregarded. The frequency of breakage was expressed as
breaks per cell (b/c) for purposes of comparison.
The reliability of cytogenetic scoring has previously
been evaluated by comparing four separate blood samples
from a respective donor with a minimum interval between
samples of 1 week.15 Using a random-effect, one-way
analysis of variance model, significance within group variation was noted, suggesting that sensitivity appeared to
be stable and representative.
Nutrient Estimates
A modified National Cancer Institute Health Habits
and History Questionnaire26-28 including food items, frequency of consumption, usual portion size, and a
restaurant use section was employed to collect usual dietary history. The estimates of nutrients were calculated by the National Cancer Institute’s algorithm.26.27
In brief, nutrient indices were obtained from the Second
National Health and Nutrition Examination Survey29
conducted from 1976 to 1980, which was based on U.S.
Department of Agriculture food composition computer
data tape,30 as well as industry and other sources. Nutrient composition values for vitamin A are based on
Revised Handbook 8 values.31 The food database includes three parts: portion sizes, food in grams, and nutrients per 100 g for each food item. Each nutrient was
calculated by the following equation: (reported food frequency x gram portion size x the nutrient contentI100 g
x seasonality factor) +loo. Nutrients were then
summed over all food and beverage items, including
restaurant foods, on the questionnaire to obtain the average intake per day. During calculation, vegetables
and fruits were adjusted for seasonal consumption by
multiplying the computed yearly intake of each food
item by the seasonal index (proportion of year during
which seasonal consumption of food occurred). The
restaurant food frequency was compared with the frequency of the corresponding food item in the main food
list, and appropriate adjustment was made before the
calculation of the estimates according to National Cancer Institute’s algorithm.26.27 Median values of each nutrient were used for subjects with missing information
on the dietary section (a total of 21 subjects including
seven cases and 14 controls were missing). A Statistical
Application System program was developed to calculate
nutrients in this study.
Statistical Analysis
Uniuariate analysis. The relationship between risk
of head and neck cancer and putative risk factors such as
mutagen hypersensitivity; intake of dietary vitamins C, E,
and carotenoids; cigarette smoking; alcohol drinking; and
body mass index (BMI) were measured using the odds ratios (OR) and their 95% confidence interval (95% CI), derived from logistic regression analysis.32 Variables of nutritional vitamin intakes were divided into quartiles
according to their distribution. Dummy variables were
employed t o estimate the OR for each category of exposure
in logistic regression analysis. The presence of a trend for
ordered variables was initially assessed by Mantel-Haenszel extension methods and then performed by assigning
the score j to the jth exposure level of a categorical variable and treating it as a continuous variable in unconditional logistic regression.33
Schantz et al.: Genetic Susceptibilityto Cancer
767
Interaction assessment. Stratified analysis was
used to assess possible additive interaction and, if no interaction was found, to control for potential confounding
effects and to obtain a summary statistics by using the
Mantel-Haenszel method.33 The test of significance for additive interaction between two risk factors was performed
by methods described by Rothman34 and Daya1.35 The
data were stratified and compared between various subgroups against one standard reference group. The standard reference group was usually selected to be the group
with minimal level of exposure on all risk factors for head
and neck cancer.36 For example, to assess the additive interaction between cigarette smoking and mutagen sensitivity on cancer risk, cases and controls were stratified
into four groups according to smoking (yeslno) and mutagen sensitivity ( 4and 21):nonsmoking and mutagen sensitivity d, smoking and mutagen sensitivity d,nonsmoking and mutagen sensitivity 21, and smoking and
mutagen sensitivity 21. For each of four combinations of
smoking and mutagen sensitivity, the odds of disease can
be calculated by dividing the number of cases over the
number of controls. To calculate the OR for each category,
a reference group with minimum exposure was chosen
(nonsmoking and mutagen sensitivity <l); then the odds
of disease of each group was compared with the odds of
disease of the reference group. The synergy index (S) was
calculated and a chi-squared test with one degree of freedom was employed to test the significance of synergy.34~35
Multiplicative interaction was assessed by unconditional
logistic regression model for mutagen sensitivity and
other risk factors that were significant in univariate
analysis. A product term of mutagen sensitivity and each
potential risk factor was created and introduced into the
multivariate model with both independent variables.
Multivariate analysis. All variables that were significant in univariate analysis were entered into a multivariate logistic model after controlling for potential confounding effects such as age, sex, race, and education.
Odds ratios and their 95%CIS were estimated and trend
tests were performed.
RESULTS
Demographic characteristics of both the cases
and controls are shown in Table I. Overall, no differences in age, race, sex, or place of birth could be identified between cases and controls.The mean ages were
60.9 years (SD k12.37) for cases and 58.4 years (SD
43.63) for controls (Student’s t-test, P > 0.05). Similar distributions were found for cases and controls for
gender and race. The cancer population was less
likely to be college educated (P c 0.01). Likewise, the
median income of the cancer patients was lower than
the controls, but this did not reach statistical difference (P = 0.05). The above data suggests that cases
and controls were comparable in terms of age, sex,
and race in this study.
The association between selected factors and the
risk of head and neck cancer was analyzed after controlling for potential confounding effects such as age,
sex, and race (Table 11).Current tobacco use was associated with a significantly increased risk of head
Laryngoscope 107:June 1997
768
and neck cancer (OR = 6.22; 95% CI = 3.55 to 12.35)
(P = 0.0001). A strong dose-response relationship
was observed between cigarettes per day and the
risk of head and neck cancer: the ORs were 2.19
(95% CI = 1.23 to 3.92) for those who smoked one to
20 cigarettes per day, and 4.62 (95% CI = 2.46 to
8.69) for those smoked more than 20 cigarette per
day (trend test, P = 0.0001). Of those who had
stopped using tobacco for at least 5 years prior to diagnosis, the OR of head and neck cancer was only
marginally elevated (OR = 1.16; 95% CI = 0.62 to
2.17 (P > 0.05).The median duration of smoking cessation for cancer patients was 3 years (range, 1to 66
years) and that of smoking cessation for controls was
16.5 years (range, 1 to 53 years). Passive smoking
was identified to be associated with an increased
risk of head and neck cancer (OR = 3.14; 95% CI =
1.45 to 6.76).The use of alcohol was associated with
increased risk of head and neck cancer in only those
individuals with highest level of alcohol use (i.e.,
>lo0 drinks per month (OR = 6.22; 95% CI = 2.83 to
13.65) (trend test, P = 0.001). Increased body
weight and BMI were significantly associated with a
decreased risk of head and neck cancer (trend tests,
P = 0.0005 and P = 0.0302, respectively) (Table 11).
Dietary nutrients were examined after controlling for age, sex, race, and calorie intake. No obvious
association was identified for total dietary protein,
fat, or carbohydrates after controlling for age, sex,
race, and total intake of calories. High intakes of vitamins C and E were related to a decreased risk
(trend tests, P = 0.0049 and P = 0.0093, respectively). Although dietary beta-carotene was not associated, high intake of other specific carotenoids
such as cryptoxanthin and lycopene were related
to a decreased risk (trend tests, P = 0.0004 and
P = 0.0379, respectively) (Table 111).No significant
association was observed between vitamin supplement (A, C, and E) and risk of head and neck cancer,
although the ORs for total intake of vitamins C and
E were protective. Additional analysis on food items
showed that risk of head and neck cancer was significantly increased with high consumption of the
following food items: liverwurst, eggs, ice cream,
beef, liver, pork, and fried chicken; the risk was decreased with high intake of fruits and vegetables
such as apples, tomatoes, and green salad (data not
shown).
An analysis was performed of mutagen sensitivity as a risk factor for head and neck cancer development. Table IV provides risk estimates after
adjusting for age, sex, and race. Various cutoff points
were examined including the simple division of the
control population into approximately four equal
groups. In each instance the highest risk was associated with a breaks per cell (b/c) value 21 (OR =
5.99; 95% CI = 3 to 11.99). Previous case-control
studies have used ~ 0 . 8b/c as a cutoff value between
mutagen-sensitive and mutagen-nonsensitive indiSchantz et al.: Genetic Susceptibility to Cancer
TABLE I.
Demographic Characteristics of Cases and Controls.
Factors
Cases (n = 167)
No.
Yo
Controls (n = 177)
No.
%
P value
Age (Y)
c40
40-49
50-59
60-69
270
*
Mean SD
Sex
Male
Female
Race
White
Non-white
Education
<High school
College
>College
Place of birth
United States
Elsewhere
Religion
Catholic
Jewish
Baptist
Other Protestant
Seventh-Day
Adventists
Other religions
None
Family income ($/year)
~$13,000
$13,000-$22,999
$23,000-$32,999
$33,000-$42,999
$43,000-$52,999
$53,000-$62,999
1$63,000
Mutagen sensitivity test
No
Yes
Site
Oral cavity
Pharynx
Larynx
Nasal cavity
Unknown
Laryngoscope 107: June 1997
7
22
48
50
43
60.9 i 12.37
4.1
12.9
28.2
29.4
25.3
5.84i 13.63
19
24
43
56
35
10.7
13.6
24.3
31.6
19.8
0.14
107
57
65.2
34.8
111
63
63.8
36.2
0.781
146
19
88.5
11.5
157
16
90.8
9.2
0.494
102
47
18
61.1
28.1
10.8
49
90
39
27.5
50.6
21.9
0
139
31
81.8
18.2
156
23
87.2
12.8
0.164
104
15
8
30
62.3
9
4.8
18
91
38
4
32
51.1
21.4
2.3
18
1
7
2
0.6
4.2
1.2
1
6
6
0.6
3.4
3.4
0.03
13
17
27
18
12
13
34
9.7
12.7
20.2
13.4
9
9.7
25.4
8
9
21
20
21
19
57
5.2
5.8
13.6
12.9
13.6
12.3
36.8
0.053
76
91
45.5
54.5
46
131
79
25
42
1
3
52.7
16.7
28
0.7
2
26
74
Schantz et al.: Genetic Susceptibility to Cancer
769
TABLE (I.
Analysis of Selected Risk Variables.
Variables
Cigarette smoking
No
Yes
Quit
Current+
No./day
1-20
>20
Cases (n)
Controls (n)
Odds Ratios'
95% CI
27
138
41
97
62
112
72
40
1
2.9
1.16
6.22
1.7-4.95
0.62-2.17
3.55-12.35
69
67
72
35
2.19
4.62
P = 0.0001
1.23-3.92
2.46-8.69
20
68
42
46
40
15
1.05
3.89
6.72
P = 0.0001
0.51-2.16
2.1-7.19
3.06-1 4.7
10
156
29
145
27
41
33
61
36
63
51
16
1
0.88
0.99
6.22
P = 0.0001
0.45-1.72
0.49-1.99
2.83-1 3.65
52
67
26
18
40
41
42
40
1
1.17
0.43
0.35
P = 0.0005
0.61-2.25
0.2-0.91
0.15-0.77
56
41
40
26
40
40
39
41
1
0.66
0.68
0.46
P = 0.0302
0.35-1.24
0.36-1.28
0.23-0.89
Trend test
Years of smoking
1-20
21-40
>40
Trend test
Passive smoking
No
Yes
Alcohol use (drinksho)
No
1-30
31-100
>loo
Trend test
Usual body weight (kg)
165.8
65.9-80.8
80.9-93.1
293.2
Trend test
Body mass index
523.2
23.3-25.8
25.9-29
229.1
Trend test
1
3.14
1.45-6.76
'Odds ratios were adjusted for age, sex, and race.
+Current smokers included smokers who had quit less than 5 years previously.
viduals. Using this latter cutoff point, the OR of
head and neck cancer was 3.97-fold higher in the
mutagen-sensitive population (95% CI = 2.11 to
7.47). We also divided cases and controls into three
categories: ~ 0 . 8b/c, 0.8 to e l b/c, and 2 1 b/c. The
ORs were 1.75 (95% CI = 0.75 to 4.08) for individuals with mutagen sensitivity between 0.8 and 1b/c
and 5.99 (95% CI, 3 to 11.99) for those whose mutagen sensitivity was 1 b/c or higher (trend test,
P=O.OOOl). We then compared individuals with a
mutagen sensitivity value of 1 b/c or higher with
those with a value lower than 1b/c. The OR was 4.95
(95% CI = 2.67 to 9.17).
Laryngoscope 107:June 1997
770
The associations between mutagen sensitivity
and the above-mentioned risk factors including cigarette smoking, alcohol drinking, and dietary vitamin C, E, cryptoxanthin, and lycopene intake, were
assessed separately in both cases and controls, but
no statistically significant association was observed
(Table V). These results suggest that intake of various nutrients, tobacco, and alcohol did not influence
individual mutagen sensitivity values.
Table VI shows risk estimates for additive interactions between cigarette smoking; alcohol use; and
dietary vitamin C, E, cryptoxanthin, lycopene intake;
and mutagensensitivity We used >1b/c as the cutoff
Schantz et al.: Genetic Susceptibilityto Cancer
TABLE Ill.
Analysis of Dietary Vitamin Intakes*
Variables
Odds Ratios"
Cases (n)
Controls (n)
95% CI
57
37
40
36
45
43
47
44
52
45
20
53
45
45
45
44
27
55
26
62
45
45
45
44
32
60
30
48
45
45
50
39
1
1.75
0.67
1.3
P = 0.7988
0.94-3.25
0.34-1.31
0.67-2.55
37
43
26
64
45
45
49
40
1
0.96
0.46
1.39
P = 0.6221
0.51-1.81
0.23-0.91
0.73-2.67
61
39
40
30
45
43
47
44
54
34
25
57
45
45
47
42
70
26
22
52
45
45
45
44
1
0.34
0.26
0.6
P = 0.0379
44
45
1
Vitamin C (mg)
<91.5
915141.4
141.5-175.2
>175.2
Trend test
Vitamin E (a-TE)
<5.4
5.4-7.7
7.8-8.6
28.6
Trend test
Vitamin A (IU)
<5256
5256-8622
8622-10565
210565
Trend test
Alpha carotene (pg)
<181.9
181.9-426.2
426.3-609.5
2609.5
Trend test
Beta carotene (pg)
~2064
2064-3503
3503-4421
24421
Trend test
Cryptoxanthin (pg)
<61.4
61.4-1 15.3
115.3-148
2148
Trend test
Lutein (pg)
4678
1678-3333
3333-3732
>3732
Trend test
1
0.68
0.48
0.4
P = 0.0049
1
0.54
0.2
0.45
P = 0.0093
0.37-1.27
0.26-0.9
0.2-0.8
0.28-1.03
0.09-0.43
0.21-0.95
1
1.89
0.68
1.64
P = 0.6309
0.99-3.59
0.33-1.41
0.83-3.26
1
0.64
0.45
0.3
P = 0.0004
0.35-1.18
0.24-0.84
0.15-0.6
1
0.58
0.34
0.95
P = 0.5708
0.31-1.08
0.17-0.66
0.52-1.73
Lycopene (pg)
<910.7
910.7-1509.4
1509.4-1898.4
21898.4
Trend test
Retinol (pg)
<323.9
0.18-0.64
0.13-0.51
0.32-1.I 1
(Continues)
Laryngoscope 107: June 1997
Schantz et al.: Genetic Susceptibility to Cancer
771
TABLE 111. (Continued)
Analysis of Dietary Vitamin Intakes
Variables
323.9-506.9
506.9-600.4
>600.4
Cases (n)
Controls (n)
Odds Ratios*
95% CI
35
28
63
45
45
44
0.68
0.5
1.07
P = 0.9622
0.35-1.3
0.26-0.99
0.55-2.08
32
48
30
60
45
45
45
44
1
1.37
0.68
1.39
P = 0.6582
Trend test
Pro-A carotene (pg)
~2320
2320-3920
3920-4959
s4959
Trend test
0.73-2.57
0.35-1.36
0.73-2.67
'Odds ratios were adjusted for age, sex, race, and caloric intake.
value to define hypersensitivity for this analysis. In
nearly every instance an interactive effect was suggested ( S >O). The risk estimate was markedly higher
when assessing for the presence of combined risk factors than for each risk factor alone. A n additive interaction effect was identified when evaluating the combined effect of cigarette smoking and mutagen
sensitivity. The ORs were 5.34 (95% CI = 1.04 to
27.26) for individuals who had a positive test result
for mutagen sensitivity (21 b/c) but were nonsmokers,
5.05 for individuals who had a negative test result for
mutagen sensitivity (<1 b/c) but were smokers, and
24.92 (95%CI = 6.59 to 94.24) for those with a positive test result who were smokers (S = 1.41;x2 = 3.86;
P < 0.05). Individuals who both consumed large
amounts of alcohol (>lo0 drinks per month) and were
hypersensitive to mutagens had an OR of 45.33 (95%
CI = 9.25 to 222).The joint effects were also identified
for mutagen sensitivity and passive smoking (OR =
12.11; 95%CI = 3.19 to 46.031, lower dietary vitamin
C intake (OR = 15.6;95%CI = 5.56 to 43.711, lower vitamin E intake (OR = 13.69;95%CI = 4.79to 39.Q lower
cryptoxanthin (OR = 12.05; 95% CI = 4.66 to 31.12),
lower lycopene intake (OR = 8.06; 95% CI = 3.11 to
20.85), and lower BMI (OR = 11.11; 95%CI = 4.19 to
29.46).None of these fadors were identifled to have a
signiscant multiplicative interaction with mutagen
sensitivity on the risk of head and neck cancer (Table
VII).
In the multivariate risk model after including
variables that were significant in the univariate
analysis, the ORs were 4.58 (95%CI = 1.37 to 15.32)
for cigarette smoking, 7.31 (95%CI = 2.36 to 22.67)for
alcohol use, and 7.75 (95%CI = 2.95 to 20.35) for mutagen sensitivity. Lower dietary intake of vitamins
was associated with increased risk but was not statistically significantly related to the risk of head and
neck cancer (Table VII).
Laryngoscope 107: June 1997
772
DISCUSSION
The present case-control study demonstrates the
significance of a quantitative analysis of mutagen
sensitivity within head and neck cancer patients.
Specifically, our data reveal that the OR of head and
neck cancer is markedly elevated in individuals who
express sensitivity to the clastogen bleomycin. The
case-control study here confirms a previously performed case-control study that used a convenience
sample as the control population.16 In that initial
study, a subsequent study by Spitz et al.,17 and this
present study an enhanced interaction was noted between tobacco use and mutagen sensitivity. Indeed,
results among the three studies were nearly identical
with ORs of head and neck cancer of 5.05 in individuals who smoked but were not mutagen sensitive, 5.34
in those who did not use tobacco but were mutagen
sensitive, and 24.9 in those who both smoked and
were mutagen sensitive. "he O h for head and neck
cancer in the original convenience sample and in the
subsequent case-control study by Spitz et al. were
19.8 and 23, respectively.lGJ7 Unlike these previous
two studies, our analyses also adjusted for age, sex,
and race, and used a higher cutoff ratio for hypersensitivity to mutagens, namely 1 b/c as compared with
0.8 b/c.
The present case-control analysis is the first such
analysis to identify a relationship between diet, mutagen sensitivity, and head and neck cancer. Patients
in this study could be characterized by their low fruit
and vegetable consumption as compared with the
age- and sex-matched controls. The OR of head and
neck cancer increased in the group of patients with
the least consumption of these food stuffs.Similar observations were noted when calculations for specific
nutrients were derived from the food frequency questionnaire. An increased risk of head and neck cancer
was noted in individuals who had low intake of vitSchantz et al.: Genetic Susceptibility to Cancer
TABLE IV.
Mutagen Sensitivity and Risk of Head and Neck Cancer.
Variables
Cases (n)
Controls (n)
Odds Ratios*
95% CI
Mutagen sensitivity
(breakskell)
<1
21
<0.8
0.8-<1
21
35
56
22
13
56
101
30
72
29
30
1
4.95
1
1.75
5.99
P = 0.0001
11
10
14
56
34
35
32
30
1
0.89
1.55
5.62
P = 0.0001
22
69
72
59
11
10
14
56
34
35
30
32
1
0.89
1.66
5.21
P = 0.0001
0.32-2.44
0.63-4.38
2.19-12.38
10
11
14
56
23
40
38
30
1
.
0.63
0.9
3.99
P = 0.0001
0.22-1.78
0.33-2.47
1.56-1 0.16
Trend test
Quartile (1 fixed)
<0.58
0.58-0.76
0.77-0.99
tl
Trend test
Mutagen sensitivity
(breakskell)
<0.8
20.8
1
3.97
2.67-9.17
0.75-4.08
3-1 1.99
0.32-2.46
0.59-4.08
2.35-13.44
2.11-7.47
Quartile
10.56
>O .56-<0.76
>0.76-50.96
>0.96
Trend test
Quartile
<0.52
20.52-<0.76
20.76-<1
21
Trend test
*Odds ratios were adjusted for age, sex, and race.
amins C and E as well as low intake of other lessunderstood nutrients such as cryptoxanthin. This
latter agent is a retinoid precursor that functions to
inhibit head and neck cancer cells by mechanisms
distinct from beta-carotene or retinol palmitate.37
Free Radical Oxygen Damage and Its Repair
The findings of this case-control study, as well
as previous in vitro and epidemiologic studies, point
to the relevance of free radical oxygen (FRO) damage and its repair as important determinants of
head and neck carcinogenesis.l,4,23,24,38
FRO is generated by a variety of mechanisms, including both
normal endogenous metabolism and various carcinogenic exposures (Fig. 1L39-43 Tobacco, for instance, has been noted to contain over 5000 compounds, many of which have oxidant capacity.42.44
Three important classes of FRO are produced on a
daily basis within an individual and have the capacity to induce nearly 20,000 potentially genotoxic
lesions per day: superoxides ( 0 2 - ) , singlet oxygen
Laryngoscope 107:June 1997
and hydroxyl radicals (OH1.40-44 The majority
are generated during the sequential reduction of
oxygen to water. Oxidative damage to DNA mediated by these molecules is complex and involves
both direct and indirect mechanisms.38 The latter
may include inactivation of target enzymes that are
involved in the synthesis of DNA precursor molecules. Direct FRO-induced DNA damage includes
single- and double-strand breaks and the induction
of apyrimidinic sites. It also includes the generation
of numerous types of DNA adducts and resultant base
modifications such as thymine glycol, 5-hydroxymethyluracil, and 8-hydroxy-2-deoxyguanosine.38
The ability to effectively repair FRO-induced genetic
damage would thus be critical to the prevention of cell
death andor mutational events.
(l02),
Relevant to our understanding of head and neck
carcinogenesis, as well as to the interpretation of
our present case-control results, numerous reports
have demonstrated the capacity for tobacco-combustion products to induce FRO damage (reviewed in
Schantz et al.: Genetic Susceptibility to Cancer
773
TABLE V.
Distribution of Mutagen Sensitivity by Selected Factors and Disease Status.
BreaksKell
Cases
Controls
Smoking status, n (%)
Current
c0.8
0.k1
21
Former
Nonsmoker
2 (22.2)
6 (24)
1 (11.1)
3 (12)
16 (64)
6 (66.7)
x 2 = 0.463; P = 0.977
13 (24.1)
9 (16.7)
32 (59.2)
Current
Former
Nonsmoker
18 (58.1)
8 (25.8)
5 (16.1)
30 (55.6)
9 (16.7)
15 (27.7)
x 2 = 2.99; P = 0.56
21 (48.8)
12 (27.9)
10 (23.3)
1-20
None
34 (61.8)
9 (16.4)
12 (21.8)
x 2 = 2.61 ; P = 0.626
21 (48.8)
12 (27.9)
lO(23.3)
Cigarettedday, n (YO)
1-20
120
<0.8
0.8-4
21
None
>20
13 (50)
7 (26.9)
6 (23.1)
8 (20.5)
2 (22.2)
1 (11.1)
6 (15.4)
25 (64.1)
6 (66.7)
~2 = 0.694; P = 0.952
11 (25.5)
6 (15)
23 (57.5)
Alcohol drinking, n (Yo)
Yes
<0.8
0.&<1
<1
No
11 (31.4)
8 (22.9)
36 (45.7)
x2
Yes
No
8 (615)
58 (53.2)
3 (23.1)
24 (22)
2 (15.4)
27 (24.8)
x 2 = 0.381; P = 0.826
11 (21.2)
5 (9.6)
36 (69.2)
= 5.26; P = 0.072
Dietary vitamin C intake, n (“/.)
Low
c0.8
0.841
21
Low
High
15 (26.8)
10 (17.9)
31 (55.4)
x2
7 (20)
3 (8.6)
25 (71.4)
= 2.61 ; P = 0.271
High
36 (49.3)
19 (26)
18 (24.7)
= 2.31 ; P = 0.316
36 (62.1)
10 (17.2)
12 (20.7)
x’
Dietary vitamin E intake, n (7.)
Low
<0.8
0.841
21
Low
High
17 (29.3)
9 (15.5)
34 (55.2)
x2
5 (15.2)
4 (12.1)
24 (72.7)
= 2.97; P = 0.227
High
36 (56.2)
36 (53.7)
14 (21.9)
15 (22.4)
14 (21.9)
16 (23.9)
x 2 = 0.099; P = 0.952
Dietary cryptoxanthin intake, n (Yo)
Low
c0.8
0.841
t l
Low
High
14 (24.6)
9 (15.8)
34 (59.7)
x2
8 (23.5)
4 (11.8)
22 (64.7)
= 0.339; P = 0.844
High
36 (60)
36 (50.7)
10 (16.7)
19 (26.8)
14 (23.3)
16 (22.5)
x 2 = 2.02; P = 0.365
Dietary lycopene intake, n (YO)
Low
c0.8
0.8-4
21
High
15 (28.3)
8 (15.1)
30 (56.6)
x2
Laryngoscope 107: June 1997
774
7 (18.4)
5 (13.2)
26 (68.4)
= 1.45; P = 0.483
Low
High
34 (52.3)
38 (57.6)
15 (22.7)
14 (215)
13 (19.7)
17 (26.2)
x 2 = 0.78; P = 0.676
Schantz et al.: Genetic Susceptibility to Cancer
TABLE VI.
21 )*
Risk Estimates for Additive Interactions Between Cigarette Smoking, Alcohol Use, and Mutagen Hypersensitivity (4,
Variables
Cigarette smoking
No
No
Yes
Yes
Odds Ratio?
Mutagen Sensitivity
Cases (n)
Controls (n)
95% CI
No
Yes
No
Yes
3
6
31
48
S = 1.41
1
5.34
5.05
24.92
P < 0.05
1.04-27.26
1.4-1 8.24
6.59-94.24
'x
33
10
65
20
= 3.86
Passive smoking
No
No
Yes
Yes
No
Yes
No
Yes
3
3
32
51
s = 1.21
18
5
81
25
x2 = 3.21
1
3.67
2.55
12.11
P > 0.05
0.55-24.6
0.69-9.38
3.19-46.03
Alcohol use
No
No
Yes
Yes
No
Yes
No
Yes
16
36
19
16
S = 5.13
1
6.74
11.3
45.33
P > 0.05
3.06-14.84
4.23-30.17
9.25-222.1
'x
82
27
11
2
= 0.72
Vitamin C intake
High
High
Low
Low
No
Yes
No
Yes
10
25
25
31
S = 0.83
55
18
46
12
x2 = 0.71
1
8.33
4.47
15.6
P > 0.05
3.21-21.64
1.73-1 1.57
5.56-43.71
Vitamin E intake
High
High
Low
Low
No
Yes
No
Yes
9
24
26
32
S = 0.44
51
16
50
14
x2 = 0.25
1
8.44
3.89
13.69
P > 0.05
3.15-22.61
1.46-1 0.41
4.79-39.1
Cryptoxanthin intake
High
High
Low
Low
No
Yes
No
Yes
12
22
23
34
S = 0.77
55
16
46
14
x2 = 0.73
1
6.08
2.97
12.05
P > 0.05
2.38-15.53
1.22-7.23
4.66-31.12
Lycopene intake
High
High
Low
Low
No
Yes
No
Yes
12
26
23
30
S = 0.59
1
5.52
1.68
8.06
P > 0.05
2.21-13.77
0.71-3.96
3.11-20.85
'x
48
17
53
13
= 0.42
1.82-16.12
1.62-10.87
6.97-56.67
Usual body weight
High
High
Low
Low
No
Yes
No
Yes
8
12
22
44
S = 1.65
51
14
38
15
x2 = 3.01
1
5.43
4.2
19.87
P > 0.05
Body mass index
High
No
9
48
1
(Continues)
Laryngoscope 107: June 1997
Schantz et al.: Genetic Susceptibility to Cancer
775
TABLE VI. (Continued)
Risk Estimates for Additive Interactions Between Cigarette Smoking, Alcohol Use, and Mutagen Hypersensitivity ( 4 ,21):
Variables
Mutagen Sensitivity
Cases (n)
High
Low
Low
Yes
21
21
35
No
Yes
Controls (n)
Odds Ratiot
14
39
15
S = 0.48
'x
7.4
3.02
11.11
= 0.29
95% CI
2.70-20.27
1.22-7.49
4.19-29.46
P > 0.05
'The cut-point for low/high for vitamins C and E, usual body weights and body mass index was the median value of the control group.
'For smoking, passive smoking, and alcohol use, odds ratios were adjusted for age, sex, and race; for nutritional factors and body mass index, odds ratios
were adjusted for age, sex, race, and total calorie intake.
Pryor and Stone42). The active oxygen within cigarette smoke condensate is generated mostly from
polyphenols such as catechols and catechol-methyl
derivatives, as well as hydroquinone, each of which
occurs in abundance in cigarette smoke. Concrete
evidence for the ability of cigarette smoke condensate to induce DNA single-strand breaks was
demonstrated in the mid-1980s. Nakayama et a1.45
used the alkaline elution technique to measure DNA
fragments. Large amounts of FRO could be generated by trapping tobacco smoke within phosphatebuffered saline. Exposure of DNA to the FRO-containing solution led to a measurable increase in
DNA fragmentation. In support of the role of FRO
as the damaging agent, DNA fragmentation could
be inhibited by exposing the DNA to naturally occurring enzymes that have as their sole function the
conversion of FRO to inactive species. Weitberg and
TABLE VII.
Risk Estimates for Multiplicative Interactions Between Cigarette Smoking,
Alcohol Use, and Mutagen Sensitivity (el,>l)."
Variables
Odds Ratios
P value
95% CI
Smoking (yes vs. no)
Mutagen sensitivity
Interaction
5.05
5.34
0.93
1.4-1 8.24
1.04-27.26
0.16-5.42
Passive smoking (yes vs. no)
Mutagen sensitivity
Interaction
2.55
3.67
1.3
0.69-9.38
0.55-24.6
0.18-9.59
0.1606
0.18
0.7995
Drinking (100/movs. others)
Mutagen sensitivity
Interaction
11.3
6.74
0.6
4.23-30.17
3.06-14.84
0.1-3.75
0.0001
0.0001
0.5808
Vitamin C intake (low vs. high)
Mutagen sensitivity
Interaction
4.47
8.33
0.42
1.73-11.57
3.21-21.64
0.12-1.5
0.002
0.0001
0.182
Vitamin E intake (low vs. high)
Mutagen sensitivity
Interaction
3.89
8.44
0.42
1.46-10.41
3.15-22.61
0.12-1.51
0.0001
0.1819
Cryptoxanthin intake (low vs. high)
Mutagen sensitivity
Interaction
2.97
6.08
0.67
1.22-7.23
2.38-1 5.53
0.19-2.33
0.0168
0.0002
0.5271
Lycopene intake (low vs. high)
Mutagen sensitivity
Interaction
1.68
5.52
0.87
0.71-3.96
2.21-13.77
0.25-2.99
0.2339
0.0003
0.8217
Usual body weight (low vs. high)
Mutagen sensitivity
Interaction
4.2
5.43
0.87
1.62-10.87
1.83-16.12
0.22-3.42
0.0031
0.0023
0.8442
Body mass index (low vs. high)
Mutagen sensitivity
Interaction
3.02
7.4
0.5
1.22-7.49
2.70-20.27
0.14-1.83
0.0172
0.0001
0.2925
,
0.0136
0.0442
0.9319
0.0068
*For dietary vitamin intake, age, sex, race, and total calories were adjusted. For all other
factors, age, sex, and race were controlled.
Laryngoscope 107: June 1997
776
Schantz et al.: Genetic Susceptibility to Cancer
TABLE VIII.
Multivariate Logistic Regression Analysis of Risk Factors for Upper
Aerodigestive Tract Cancer.'
Variable
Cigarette smoking (yes vs. no)
Passive smoking (yes vs. no)
Alcohol use (>lo0drinks/mo vs. others)
Vitamin C intake (low vs. high)
Vitamin E intake (low vs. high)
Cryptoxanthin intake (low vs. high)
Lycopene intake (low vs. high)
Body mass index (low vs. high)
Mutagen sensitivity (21 vs. 4)
Age (Y)
Sex (M/F)
Race (Nonwhite vs. white)
Education*
Family Incomes
Odds Ratio*
95% CI
4.58
6.06
7.31
2.48
2.02
1.17
1.14
2.09
7.75
1.03
1.87
2.84
1.03
0.8
1.37-15.32
0.91-40.22
2.36-22.67
0.71-8.71
0.65-6.28
0.39-3.52
0.42-3.11
0.87-5.05
2.95-20.35
0.99-1.07
0.66-5.31
0.56-14.27
0.50-2.11
0.62-1.05
*Total calorie intake was adjusted in the analysis.
*Odds ratios were estimated when all variables were included in a multiple logistic
regression model.
*Education was defined as grade school = 1; some high school = 2; high school graduate
= 3;technicalhrocationalschool certificate = 4;some college = 5;college graduate = 6;
postgraduate degree = 7.
§Incomewas defined as c$13,000/y = 1; $13,000-22,999= 2;$23,000-32,999= 3;
$33,00042,999= 4;$43,000-52,999= 5;$53,00042,999
= 6;>$63,000= 7.
Corvese46 extended these observations by demonstrating the ability of tobacco-specific nitrosamines
to potentiate FRO production and double-strand
DNA breakage. Similar to findings of Nakayama et
al., Weitberg and Corvese showed that free radical
scavengers abrogated the nitrosamine-induced
damage.
The complexity of DNA repair of FRO damage
is readily apparent. A multiplicity of genes and gene
products are involved. In a recent review of the subject, Pryor and Stone42 point to two principle mechanisms in which aberrant repair may contribute to
mutations following FRO-induced strand breakage.
CELL
MEMBRANE
Error-prone DNA ligase activity may join DNA
strands across gaps following excision of altered
bases. When such repair occurs across strand breaks
accompanied by base release, a deletional mutation
would result. The second mechanism relates to base
misincorporation by aberrant polymerase activity in
the attempt to restore normal sequences in damaged genes.
Bleomycin and Free Radical Oxygen
Also pertinent to the interpretation of this casecontrol study as well as to the understanding of
head and neck carcinogenesis, numerous studies
CYTOPLASM
NUCLEUS
Selenium
Fig. 1. The generation of free radical oxygen and its modulation by cellular constituents. Free radicals are generated
from both endogenous and exogenous
sources. Constituentswithin the extracelMar space, cell membrane, or cellular
cytoplasm can either block (-) or enhance (+) free radical carcinogenic potential. BAP = benzo(a)pyrene; HQ = hydroquinones; CC = catechols; Fe = iron;
TSN = tobacco-specific nitrosamines,
GSTu = glutathione-S-transferase mu;
SOD = superoxides dismutase; CAT =
catalase.
Laryngoscope 107: June 1997
Schantz et al.: Genetic Susceptibility to Cancer
777
have demonstrated that bleomycin induces chromosomal breakage by means of generation of free oxygen radicals.8-10,47-49On initial entry into the cell,
bleomycin complexes with ferrous iron and molecular 0 2 . The complex intercalates with DNA, principally between G-T and G-C sequences, leading to
hydrogen abstraction from deoxyribose and thereby
leaving a free radical. The latter combines with 02,
which generates a peroxyl radical high-energy state,
Similar to free radical interactions from other
sources such as radiation or tobacco exposure, energy induced in this manner is decomposed through
strand breakage. It is also relevant that bleomycin
can produce 8-hydroxyguanine in DNA with a yield
approximating %OO that of strand cleavage.9 As reported by Sikic,g however, no other type of base damage was identified. Tobacco contains a broad array of
mutagenic compounds, each capable of interacting
with DNA in a characteristic manner. Bleomycin
sensitivity represents only one of several mechanisms of tobacco-induced carcinogenesis. It is, however, reflective of a very critical process (i.e., FRO
damage).
Our study demonstrated also for the first time
that specific antioxidant vitamin intake (i.e., vitamin C or E) was interactive with quantitative assessment of mutagen sensitivity in defining risk
of head and neck cancer. Both vitamin intake and
mutagen sensitivity together provided more information than either factor alone. Results were consistent with previous in uitro experimentation,
which showed the protective effect of these agents
on bleomycin-induced chromosomal damage.23224
These latter in uitro studies used peripheral blood
lymphocytes harvested from head and neck cancer
patients. Lymphocytes were cultured and exposed
to bleomycin in a manner identical t o the assay
used in our case-control study. Prior to exposure
t o bleomycin, lymphocytes were preincubated for
varying periods with differing concentrations of
vitamins A, C, or E. A clear dose-response protective effect was identified.23124 Cells preincubated
for more than 2 hours with the above vitamins
showed less sensitivity to mutagens as reflected
in lower levels of chromosomal breakage. The in
uitro concentrations of vitamins C and E that
were used in the laboratory studies by Trizna et
al.24, which protected against bleomycin-induced
damage, can be achieved within the peripheral
blood and thus are biologically and clinically relevant.403e52
Alcohol and DNA Repair
The present case-control study also confirmed a
highly significant interaction between alcohol and
mutagen sensitivity. The OR of head and neck cancer
increased dramatically in those individuals who both
drank alcohol and were mutagen sensitive (OR =
Laryngoscope 107: June 1997
778
45.8; 95%CI = 9.25 to 222.1). Results are in contrast
to a recent study of Spitz et al.17 in which the odds
ratio for interaction between alcohol and mutagen
sensitivity was 5.8. One explanation for the difference may be related to methods of categorical grouping. Spitz et al. used a cutoff value of 0.8 b/c as the
definition for mutagen sensitivity as compared with
1 b/c in our study. Likewise, referent categories for
alcohol in our study were heavy drinkers (>lo0
drinks per month) versus non-heavy drinkers. Spitz
et al. used nondrinkers only as the referent group.
Our choice of each categorical grouping was associated with an increased disease OR and would account for an increased OR when used in combination
(Tables I11 and V).
The potential interactive effect between mutagen sensitivity and alcohol demonstrated in the present case-control analysis suggests a mechanism by
which alcohol may contribute to head and neck carcinogenesis, specifically the inhibition of DNA repair16,53-56 Hsu et a1.53, and Hsu and Furlong 54 tested
this hypothesis directly using the bleomycin assay
and immortalized lymphocytes obtained from head
and neck cancer patients. Hsu et al.53 noted that coincubation of lymphocytes with alcohol increased
bleomycin-induced chromosomal breakage in a dosedependent manner. Kumano and Kajii55 showed that
ethanol-enhanced aphidocolin induced fragile sites in
human lymphocyte cultures. Aphidocolin functions to
inhibit DNA repair,57 Relevant to our case-control results, Lin et al.56 examined the effect of alcohol on
bleomycin-induced damage in Chinese hamster
ovary cells and noted enhanced levels of chromosomal breakage when alcohol was applied simultaneously with bleomycin as compared with bleomycin
alone. Pretreatment with alcohol had no effect on
chromosomal breakage, indicating that alcohol had
no direct clastogenic effect. Lin et al.,56 however,
noted that multiple mechanisms may account for
the synergistic effect between bleomycin and alcohol, inducing alcohol-mediated increased cellular
membrane permeability, alterations of bleomycin
metabolizing enzymes, and the inhibition of DNA
repair. Hsu and Furlong 54 provided supportive evidence for the capacity of alcohol t o inhibit DNA repair. The latter authors performed a kinetic analysis of repair of bleomycin-induced chromosomal
damage and noted that by 5 hours after bleomycin
exposure, breaks were repaired to a level approximating spontaneous breakage and repair. In a subsequent set of experiments, cells were washed following bleomycin exposure and then reincubated
in bleomycin-free media containing varying concentrations of ethanol. Marked inhibition of repair
occurred only in a 2%ethanol solution. The inhibition of repair could be reversed by removal of the
ethanol. The data provide direct evidence for alcohol's
inhibition of DNA repair and provides a basis for interpreting results of our case-control study and othSchantz et al.: Genetic Susceptibility to Cancer
ers regarding the interaction between alcohol and
mutagen sensitivity in contributing to head and neck
cancer risk.54
As previously discussed by Hsu et a1.53 and Hsu
and Furlong,54 several mechanisms could account for
this potentiation effect, which are independent of
DNA repair. Alcohol may increase the permeability of
the cellular membrane as well as increase the rate of
cellular movement through the G2 phase into mitosis.
Caffeine has been shown to function through this latter mechanism. The normal response to DNA damage by cells in the G2 phase is cell cycle arrest so as
to allow repair. Chromosomal damage in mitosis
would be more apparent in cells that were pharmacologically prevented from the adaptive G2-phase arrest response to clastogens. Multiple mechanisms
may account for mutagen sensitivity. Indeed, the expression of mutagen sensitivity in the head and neck
cancer population is undoubtedly a reflection of a
combination of events.
CONCLUSION
This thesis has attempted to extend our knowledge of genetic susceptibility to head and neck
carcinogenesis by performing a case-control epidemiologic investigation that incorporated the mutagen-sensitivity assay, a measure of DNA repair capacities. It is apparent that our understanding of
heritable factors contributing to head and neck carcinogenesis is in its infancy. Current perceptions
will undoubtedly change in time. It is highly likely,
however, that we are dealing with a polygenic model
of inheritance involving numerous contributing
DNA polymorphisms. The effects of modulating carcinogen metabolism, racial and gender differences,
inborn alterations in nutrient metabolism such as
iron storage, as well as DNA repair are most likely
interactive. As a result, the relative risk of disease
provided by any single genetic measure of head and
neck cancer will be limited.
We also conclude that a central feature of head
and neck carcinogenesis involves FRO damage, including its generation, promotion, and dissolution.
This may be fundamental to our understanding of
disease not only in patients who smoke, but also in
those who avoid such exposures, given that FRO is
generated from multiple sources including normal
endogenous metabolism. Some type of associated
mutational event may be the same in these individuals, even though the source of FRO-induced damage is different. Foremost may be the understanding
of factors that prevent or ameliorate FRO-DNA interactions. When speaking of heritable traits, the interactions will primarily involve the role of DNA repair.
The basis of our thesis has been the bleomycin
assay, which was used to quantitate sensitivity to
Laryngoscope 107: June 1997
mutagen damage. It is concluded that the relevance
of this assay is based on its associated FRO-damaging effect and the ability of the host to repair that
damage. Thus the assay involves only one of several
mechanisms by which tobacco and other environmental exposures can induce DNA damage, albeit, a
central mechanism. Results for this study confirm
the significance of quantitative measures of mutagen sensitivity as an independent risk factor for upper aerodigestive tract cancers. The risk interaction
was suggested between mutagen sensitivity and tobacco and alcohol. Each of these factors considered
jointly provides more information than any factor
alone. The possible interaction was found between
alcohol and mutagen sensitivity, which supports the
contention that both factors work through a common mechanism (i.e., DNA repair). Reported for the
first time are the results of combined measures of
nutrient intake and bleomycin-induced chromosomal damage as a contributing factor to head and
neck cancer. Nutrients that were significant protectors in the development of disease function principally as free radical scavengers.
The test of the significance of any epidemiologic study is the degree to which it influences future efforts involving both the basic and clinical
sciences. Several critical questions raised by this
study must be addressed. The data regarding the
heritable nature of mutagen sensitivity are still
limited. More extended pedigree analysis is required. Likewise, it is likely that the quantitative
measures of mutagen sensitivity within head and
neck cancer patients are a summation event (i.e.,
the end result of a multiplicity of factors such as
nutritional state of the cell, iron catabolism,
stress response, and DNA repair). Thus sensitivity within any single individual may be due to any
one of several factors that may distinguish that
individual from another mutagen-sensitive head
and neck cancer patient.
Clinical approaches must now be considered,
such as the incorporation of mutagen sensitivity
into chemopreventive trials designed to prevent second primaries. Indeed, such efforts are currently ongoing.21 Likewise, as a result of better defining
high-risk populations, new strategies of screening
are required. Investigations into the definition of
early disease, the role of behavior modification, the
development of new screening methods designed to
detect the field cancerization process, and the ability to provide health access to high risk populations
are all required.
BIBLIOGRAPHY
1. Schottenfeld D. The etiology and prevention of aerodigestive
cancers. Adu Exp Med Biol 1992;320:1-19.
2. Wynder EL, Stellman SD. Comparative epidemiology of tobacco-related cancers. Cancer Res 1977;37:4608-22.
3. de Villiers EM, Weidauer H, Otto H, zur Hauzen H. Papillo-
Schantz et al.: Genetic Susceptibilityto Cancer
779
mavirus DNA in human tongue carcinomas. Znt J Cancer
1985;36:575-8.
4. Winn DM. Diet and nutrition in the etiology of cancer. Am J
Clin Nutr 1995;61:437%45S.
5. Strauss BS. Cellular aspects of DNA repair. Adv Cancer Res
1995;45:45-105.
6. Hsu TC. Genetic instability in the human population: a working hypothesis. Hereditas 1983;98:1-11.
7. Hsu TC, Cherry LM, Samann NA. Differential mutagen-susceptibility in cultured lymphocytes of normal individuals
and cancer patients. Cancer Genet Cytogenet 1985;17:
307-13.
8. Iqbal ZM, Zohn KW, Ewig AG, Fornace AJ. Single-strand scission and repair of DNA in mammalian cells by bleomycin.
Cancer Res 1976;36:3834-8.
9. Sikic BI. Biochemical and cellular determinants of bleomycin
cytotoxicity. Cancer Surv 1986;5:82-91.
10. Vie BK, Lewis R. Genetic toxicology
-- of bleomycin. Mutat Res
i978$5:121-45.
11. Wang RY, Hsu TC, Brock WA, Liang J. DNA fragility and repair capability are separate genetic phenotypes: studies on
in t u nick translation and chromosome breakage. Znt J Oncol 1995;6:51-4.
12. Kuo MT, Hsu TC. Biochemical and cytological studies of
bleomycin actions on chromatin and chromosomes. Chromosome 1978;68:22940.
13. Schantz SP, Hsu TC. Mutagen-induced chromosome fragility
within peripheral blood lymphocytes of head and neck cancer patients. Head Neck 1989;11:337-42.
14. Hsu TC. Genetic predisposition t o cancer with special reference to mutagen sensitivity. Zn Vitro Cell Developmental
Biol 1987;23:591-603.
15. Hsu TC, Johnston DA, Cherry LM, et al. Sensitivity to genotoxic effects of bleomycin in humans: possible relationship
to environmental carcinogenesis. Znt J Cancer 1989;43:
403-9.
16. Spitz MR, Fueger JJ, Beddingfield NA, et al. Chromosome
sensitivity to bleomycin-induced mutagenesis in patients
with upper aerodigestive cancers: a case-control analysis.
Cancer Res 1989;49:4626-38.
17. Spitz MR, Fueger JJ, Halabi S, et al. Mutagen sensitivity in
upper aerodigestive tract cancer. Cancer Epidemiol Biomarkers Prev 1993;2:329-33.
18. Schantz SP, Hsu TC, Ainslie N, Moser RP. Young adults with
head and neck cancer express increased susceptibility
to mutagen-induced chromosomal damage. JAMA 1989;
262~3313-5.
19. Cloos J, Braakhuis BJ, Steen I, et al. Increased mutagen sensitivity in head and neck squamous cell carcinoma patients,
particularly those with multiple primary tumors. Znt J
Cancer 1994;56:816-9.
20. Li AT, Wang TD, Yang RT. Pingyangomycin-induced chromosome damage in lymphocytes of laryngeal cancer patients
and healthy control subjects. Head Neck 1994;16:510.
21. Schantz SP, Spitz MR, Hsu TC. Mutagen sensitivity in patients with head and neck cancers: a biologic marker for
risk of multiple primary malignancies. J Natl Cancer Znst
1990;82:1773-5.
22. Spitz MR, Hoque A, Trizna Z, et al. Mutagen sensitivity as a
risk factor for second malignant tumors following malignancies of the upper aerodigestive
tract. J Natl Cancer Znst
1994;86:1681-4:
23. Trizna Z, Schantz SP, Hsu TC. Effects of N-acetyl-L-cysteine
and ascorbic acid on mutagen-induced chromosomal sensitivity in head and cancer patients. Am J Surg 1991;162:
294-8.
24. Trizna Z, Schantz SP, Lee JJ, et al. In vitro protective effects
of chemopreventive agents against bleomycin-induced
genotoxicity in lymphoblastoid cell lines and peripheral
blood lymphocytes of head and neck cancer patients. Cancer Detect Prevent 1993;17:575-83.
25. Spitz MR, Fueger JJ, Borrud LG, Newel1 GR. The development of a comprehensive institutional-based patient risk
Laryngoscope 107: June 1997
780
evaluation program. Part I. Development, content, and
data management. Am J Prev Med 1988;4:183-7.
26. Block G, Dresser CM, Hartman AM, et al. Nutritional source
in the American diet: quantitative data from the NHANES
I1 Survey. Part I. Vitamins and minerals. Am J Epidemiol
1985;122:13-26.
27. Block G. Dresser CM. Hartman AM. et al. Nutrient sources in
the k e r i c a n diet:’quantitative data from the NHANES I1
Survey. Part 11. Macronutrients and fats. Am J Epidemiol
1985;122:27-40.
28. Block G. A review of validations of dietary assessment methods. Am J Epidemiol 1987;126(5):796-802.
29. National Health and Nutrition Examination Survey I1
(NHANES 11).Hyattsville, MD: National Center for Health
Statistics, 1976-1980.
30. Public use d a t a tape documentation. Model gram and nutrient composition. Hyattsville, MD: National Center
for Health Statistics, 1982 (computer tapes 5702 and
5703).
31. US Department of Agriculture. Nutrient database for standard reference: expansion of data published in Composition of Foods-Raw, Processed, Prepared. Revisions of
Agriculture Handbook No. 8. Hyattsville, MD: US Department of Agriculture, May 1988 (computer tape).
32. Breslow NE, Day NE, eds. Statistical Methods in Cancer
Research. Vol 1. The Analysis of Case-Control Studies.
Lyon, France: International Agency for Research on Cancer, 1980 (LARC scientific publication no. 32).
33. Mantel N, Haenszel W. Statistical aspects of the analysis of
data from retrospective studies of disease. J Natl Cancer
Znst 1959;22:719-48.
34. Rothman KJ. Synergy and antagonism in cause-effect relationships. Am J Epidemiol 1974;99:385-8.
35. Dayal HH. Additive excess risk model for epidemiologic interaction in retrospective studies. J Chronic Dis 1980;33;
653-60.
36. Schlesselman JJ, ed. Case-Control Studies: Design, Conduct,
Analysis. New York,.Oxford University Press, 1982:63-68.
37. Krinsky NI. Actions of carotenoids i n biological systems.
Annu Rev Nutr 1993;13:561-87.
38. Joenje H. Genetic toxicology of oxygen. Mutat Res 1989;219:
193-208.
39. Buettner GR. The pecking order of free radicals and antioxidants. Arch Biochem Biophys 1993;300:535.
40. Halliwell B. Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 1992;119:
598-620.
41. Mullaart E, Lohman PHM, Berendz F, Vijg J. DNA damage:
metabolism and aging. Mutat Res 1990;237:189-210.
42. Pryor WA, Stone K. Oxidants in cigarette smoke: radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann N Y
Acad Sci 1993;686:12-27.
43. Cheng KC, Loeb LA. Genomic instability and tumor progression: mechanistic considerations. Adv Cancer Res
1993;60:121-56.
44. Hoffmann D, and Hecht SS. Advances in tobacco carcinogenesis. In: Grover P, ed. Handbook of Experimental Pharmacology. New York: Spinger Verlag, 1989.
45. Nakayama T, Kaneko M, Kodama M, Nagata C. Cigarette
smoke induces DNA single-strand breaks in cells. Nature
1985;314:462-4.
46. Weitberg AB, Corvese D. Oxygen radicals potentiate the genetic toxicity of tobacco-specific nitrosamines. Clin Genet
1993;43:88-91.
47. Dorr RT. Bleomycin pharmacology: mechanism of action and
resistance and clinical pharmacokinetics. Semin Oncol
1992;19(S5):3-8.
48. Vorechovsky I, Munzarova M, Lokaj J. Increased bleomycininduced chromosome damage in lymphocytes of patients
with common variable immunodeficiency indicates an involvement of chromosomal instability in their cancer predisposition. Cancer Immunol Zmmunother 1989;29:
303-6.
Schantz et al.: Genetic Susceptibility to Cancer
49. Povirk LF, Austin MJ. Genotoxicity of bleomycin. Mutat Res
1991;257:127-43.
50. Frei B et al. Reactive oxygen species and antioxidant vitamins:
mechanisms of action. Am J Med 1994;97(3A): 5s-13s.
51. Krinsky NR. Mechanism of action of biological antioxidants.
Proc Soc Exp Biol Med 1992;200:24&54.
52. Pohl H, Reidy J. Vitamin C intake influences the bleomycininduced chromosomal damage assay: implications for detection of cancer susceptibility and chromosome breakage
syndromes. Mutat Res 1989;224:247-52.
53. Hsu TC, Furlong C, Spitz MR. Ethyl alcohol as a carcinogen
with special reference to the aerodigestive tract: a cytogenetic study. Anticancer Res 199l;ll: 1097-102.
54. Hsu TC, Furlong C. The role of ethanol in oncogenesis of the
upper aerodigestive tract: inhibition of DNA repair. Anticancer Res 1991;11:1995-8.
55. Kumano A, Kajii T Synergisticeffect of aphidmlin and ethanol on
the indudion of common fiagde sites.Hum Genet 1987;75:7543.
56. Lin YC, Ho IC, Lee TC. Ethanol and acetaldehyde potentiate
the clastogenicity of ultraviolet light, methyl methanesulfonate, mitomycin C, and bleomycin in Chinese hamster
ovary cells. Mutat Res 1989;216:93-9.
57. Hsu TC, Ramkissoon D, Furlong C. Differential susceptibility
to a mutagen among human individuals: synergistic effect
on chromosome damage between bleomycin and aphidiColin. Anticancer Res 1986;6:1171-6.
The Robert A Jahrsdoerfer Lectureship
The University of Texas Health Science Center
Department of Otolaryngology and Division of Plastic Surgery announce The Robert A. Jahrsdoerfer
Lectureship, July 10-12,1997. The topic will be The
Role of Unilateral Atresia Repair in Congenital
Grade I11 Microtia. Guest Speakers: Burt Brent,
Laryngoscope 107: June 1997
MD; F. Fred Aguilar, MD; Paul Lambert, MD; Robert
Jahrsdoerfer, MD; Antonio De la Cruz, MD. CME
credits will be offered. For more information contact
The Craniofacial Foundation of Houston, 6410 Fannin, Suite 927, Houston, TX 77030. TEL: 713-7970085.
Schantz et al.: Genetic Susceptibility to Cancer
781