Gea News Settembre 2001 - Danni ai coltivatori di Tabacco in Umbria
Lavoro originale. Fonte: http://brownw.newsreal.com/pages/brownw/Story.nsp?story_id=22816720&ID=brownw&scategory=Smoking+%26+Health
TOBACCO HARVESTERS may suffer an acute selflimited illness known as green
tobacco sickness (GTS).1,2 The disease, characterized by vomiting, headache,
abdominal pain and cramps, salivation, and sweating,3 occurs often among
nonsmoking harvesters, particularly when they harvest wet tobacco.4 High
nicotine levels5 in dew collected from tobacco leaves and high cotinine levels
in urine of exposed workers have led investigators to hypothesize that GTS is a
form of nicotine intoxication that results from percutaneous absorption of
nicotine.6 Nicotine is an alkaloid that causes stimulation of autonomic ganglia
and the central nervous system.7 Nicotine as a free base easily crosses the
dermal barrier,8,9 and workers harvesting tobacco leaves can absorb it through
the skin. Although more than 30,000 workers are employed every year in Italy'10
in tobacco farming, a direct measure of nicotine absorption during a regular
workshift has never been reported.
To assess nicotine absorption among tobacco harvesters, we measured nicotine
and cotinine concentrations in blood and urine samples collected before, during,
and after a regular workshift. To estimate the external dose of exposure, we
applied paper pads on the skin of each worker for later nicotine analysis.
Materials and Method
Study protocol. We studied 10 healthy, nonsmoking tobacco harvesters and 5
healthy, nonsmoking unexposed controls. We measured nicotine and cotinine levels
in plasma and urine before, during, and after a regular workshift. To ascertain
carry-over effects from previous days of work, we collected baseline urine and
blood samples 3 mo after the end of the harvesting season from 6 of the 10
exposed subjects.
Study location. The study was performed in Umbria, central Italy, on October
1, 1997. This day occurred during mid-season for tobacco harvesting and in the
middle of a work week. Umbria is a region with a high prevalence of tobacco
farming. The farm that participated in the study is one of the largest in the
region, employing approximately 400 workers each year. The farm tobacco practice
is similar to that followed throughout Italy. The variety of tobacco harvested
was Mcwrite.
Study population. Twelve healthy female nonsmokers who met all the inclusion
criteria were selected through advertisements posted in the farm office. However,
after the interview and the physical exam, 1 subject was excluded because she
was pregnant and 1 could not participate because she was ill on the day of study.
The inclusion criteria were between 20 and 50 yr of age and general good
health. Exclusion criteria were history of liver or skin disease, drug or
alcohol abuse, and use of medication that might influence nicotine absorption
(e.g., nitrates) or nicotine metabolism (e.g., barbiturates). All subjects
answered a questionnaire about personal habits (previous smoking habits,
exposure to environmental tobacco smoke at home), medical and occupational
histories, and previous occurrence of symptoms of GTS. Five healthy, nonsmoking
female volunteers were selected as nonexposed subjects among the hospital staff
(research physicians).
Environmental sampling. On the day of the study, 50 ml of dew were collected
from tobacco leaves and refrigerated for subsequent nicotine assay, Leaves from
different sites of the field were collected, stored in plastic bags, and
refrigerated for later nicotine assay.
Personal passive sampling. Paper pads (5 cm^sup 2^) were applied with tape
directly on the skin of the torso of each worker. To avoid direct contamination
with dew or tobacco leaves, we applied the pads under the clothes. Pads were
applied before the beginning of the shift and removed at the end of the shift.
Following removal, pads were placed individually in closed containers and
refrigerated for subsequent nicotine analysis.
Biological sampling
Urine. Collection of urine was begun the night before the study and completed
the night after the study at 12 P.M. Five urine specimens were collected from
each subject in acidified plastic containers. We instructed exposed and
nonexposed subjects to keep containers away from tobacco and tobacco smoke.
Urine samples were brought to the lab and frozen at -70 deg C for subsequent
analysis. Three months following the end of the harvesting season, we collected
early morning urine samples from 6 of the subjects who had participated in the
earlier study.
Blood. Blood was collected by phlebotomy from the antecubital vein. The first
sample was collected before the shift; subsequent samples were collected about
every 1 and 1/2 hr during the shift. The last sample was collected 2 hr after
the shift had ended. We used the same time schedule to collect blood from the 5
nonexposed subjects. Following collection, blood samples were stored in ice,
brought to the lab, and centrifuged, and serum was stored at -70 deg C for later
analysis. Three months after the end of harvesting season, we collected one
early morning blood sample from 6 of the subjects who participated in the study.
Exposure protocol. On the day of the study, subjects wore what they normally
wore to work: rubberized apron, gloves and boots layered above cotton clothes.
Subjects began working at 7 A.M. and worked until 10.00 A.M., at which time they
paused for a 30-min breakfast break and shed protective rubberized clothes. They
resumed work at 10:30 A.M. and continued until 2 P.M. Following a 2-hr lunch
break, the subjects returned at 4.00 P.M. for the final blood sampling. Three
months after the end of harvesting season, we collected early morning blood and
morning urine samples from each of 6 of the 10 tobacco harvesters.
Control protocol. The same sampling protocol used with the tobacco harvesters
was used for the 5 healthy volunteers who worked in our hospital. We asked the
subjects to avoid exposure to environmental tobacco smoke during working hours.
Nicotine and cotinine assays. Nicotine and cotinine were determined in blood
and urine by gas chromatography-mass spectroscopy. The limit of quantitation of
the method is 0.20 ng/ml in plasma and urine.11 Nicotine from tobacco leaves and
pads was extracted by the supercritical fluid method and then assayed by
highperformance liquid chromatography.12,13
Statistical analysis. Statistical analysis was performed with SAS software (version
6.12). To evaluate the change in nicotine and cotinine measurements over time,
we performed two-way repeated measures analysis of variance (ANOVA), with
grouping by environmental tobacco smoke (ETS) exposure (present vs. absent). We
then used Tukey's modified t test to perform pairwise comparisons. To compare
tobacco harvesters with controls, we repeated the two-way repeated measures
ANOVA with grouping by subject status (harvester vs. control).
Results
On the day of the study, the atmospheric conditions, which were typical for
the area, were cold and foggy early in the morning. Tobacco leaves were covered
with dew. Later during the day, it was sunny and warm, and no visible water
remained on the leaves.
After the study, we found that 1 subject who smoked up until 1 wk before the
study had very high plasma and urine cotinine values. Her cotinine data had to
be excluded from the analysis. One other subject had problems with phlebotomy,
and only 3 samples could be drawn from her.
Characteristics of study population. The average ages of the exposed and
nonexposed group were 49 +/- 10 yr and 32 +/- 3 yr, respectively. All exposed
and nonexposed subjects were lifetime nonsmokers, except 1 individual who had
smoked up until 1 wk before the study and 1 of the nonexposed individuals who
had smoked until 1 mo before the study. Six subjects in the exposed group and 3
in the nonexposed group were exposed to ETS at home where one or morefamily
members were smokers. None of the subjects took medication that interfered with
the absorption or metabolism of nicotine.
Environmental sampling and personal passive sampling. The concentration of
nicotine in dew collected from tobacco leaves was 1.85 (mu)g/l. Tobacco leaves
had an average nicotine concentration of 19.6 mg/gm of dry matter (range =
9.74-41.61 mg/gm). Nicotine was measurable in all the personal sampling pads;
the average amount of nicotine in the pads was 5.2 (mu)g (range = 0.5-19.5 (mu)g).
Plasma nicotine. Plasma nicotine concentrations for exposed and nonexposed
individuals are shown in Figure 1. In the exposed group, nicotine concentration
increased from a preshift value of 1.85 +/- 0.46 ng/ml to an endshift value of
3.45 +/- 0.84 ng/ml (p < .01; Table 1). Nadir and peak values were observed
at 10.30 A.wt. (i.e., before the breakfast pause) and at 4 P.M. (end of work
day), respectively. Individual plots of the blood nicotine curves showed similar
results. The difference between baseline and peak nicotine concentration for
each subject is presented in Figure 2. No differences were observed between
ETS-exposed and nonETS-exposed individuals, either at baseline or at any time
point (Table 11.
Plasma nicotine values in the nonexposed group were about 6 times lower than
plasma nicotine values in the exposed group (p < .01). No trend was observed
during the sampling times. Baseline blood nicotine, measured in 6 of the exposed
subjects 3 mo after the end of the harvesting season, was 0.24 +/- 0.12 ng/ml (n
= 6); this level was significantly lower than the last value measured during the
work day (Table 1).
Plasma cotinine. Plasma cotinine concentrations in exposed and nonexposed
individuals are shown in Figure 3. No trend was observed across the shift during
exposure (p = .22). No differences were observed between ETS-exposed and
non-ETS-exposed subjects, either at baseline or at any time point (Table 1). The
average plasma cotinine concentration measured in 6 of the exposed subjects 3 mo
after the end of the harvesting season was 0.72 +/- 0.19 ng/ml (Table 1). The
difference between exposed and control individuals did not reach statistical
significance (p = .1).
Fig. 1.
Table 1.
Fig. 2.
Urine nicotine and cotinine levels. Urine nicotine and cotinine
concentrations versus time for exposed and nonexposed individuals are shown in
Figures 4 and
5. In the exposed group, there was a trend toward increasing urine nicotine
(p = .098) and cotinine levels (p = .10) during the sampling period (Table 1),
but no such trend was present in the nonexposed group. The difference between
urine nicotine and cotinine levels in the exposed versus nonexposed group was
statistically significant (p < .01 and p < .05, respectively). The average
urine nicotine and cotinine concentrations measured in 6 of the exposed subjects
3 mo following the end of the harvesting season were 14.1 +/- 11.6 ng/ml and
6.19 +/- 1.13 ng/ml, respectively. These values were significantly lower than
the last values measured during the work day (p < .05; Table 1).
Discussion
In this article, we have presented novel data on plasma levels of nicotine
during tobacco harvesting. In other studies, investigators have reported
increased urinary excretion of nicotine and cotinine following harvesting. These
studies, however, did not provide information on the time course of exposure to
nicotine during a work day. Our analysis also provided a quantitative estimate
of nicotine exposure in tobacco harvesters.
We compared nicotine and cotinine levels in nonsmoking tobacco harvesters
with nonsmoking women who did not work with tobacco. As an additional control,
we compared levels of nicotine and cotinine in a group of tobacco harvesters
while harvesting and again-3 mo after the harvest season ended. Both control
groups had extremely low levels of nicotine and cotinine, compared with the
tobacco exposure group, thus confirming that levels measured during harvesting
were a consequence of occupational exposure.
Plasma nicotine levels in the exposed group rose significantly during the day
and peaked at the end of the work day--a fact consistent with ongoing workplace
exposure. Six of the study subjects were exposed at home to ETS. Given that the
baseline value was the same in those with and without passive smoke exposure,
one can assume that all subsequent increases in nicotine levels resulted from
workplace exposure. We were, however, surprised to observe baseline nicotine
levels among all the workers. Based on the short halflife of nicotine, one would
not expect baseline levels to be explained by a carry-over effect from previous
days of work. Nor were the workers exposed to ETS immediately before the shift
commenced. Perhaps the rain gear (i.e., rubberized boots and apron) that each
worker wore early in the morning was contaminated with residual nicotine from
previous days. Indeed, baseline plasma nicotine measured at the end of
harvesting season was approximately 10 times lower than the value measured the
day of the study.
Fig. 3.
Fig. 4.
It is also interesting that the nadir value of plasma nicotine for the work
group was reached at about 10:30 A.M., and it increased after workers shed their
protective rubberized clothes. Although early in the morning the field was wet
and leaves were moist with dew, there was little nicotine absorption, which
occurred when workers shed protective clothes at a time when the leaves and
field had been dried by several hours of sunshine. This result indicates that
dermal absorption, rather than inhalation, is the main route for nicotine
absorption in the workplace; that nicotine absorption occurs, even in the
absence of visible moisture; and that rubberized clothes effectively protect
workers from absorption. The efficacy of rubberized clothing and gloves in
protecting workers from nicotine absorption has been shown in other
studies.14,15 The measurement of nicotine on personal monitoring pads also
supports this route of exposure. Nicotine inhalation cannot be excluded; however,
if inhalation is an important route, one would expect to observe some nicotine
absorption during the first hours of exposure, when dermal absorption is
negligible, because workers are protected by rubberized clothing-but inhalation
could still occur.
Fig. 5.
Plasma cotinine levels in exposed workers were about 50 times higher than in
nonexposed workers. The values were rather stable during the workshift (i.e.,
averaging about 15 ng/ml). A flat cotinine curve reflected the long plasma
half-life of cotinine (i.e., approximately 20 hr). Urine cotinine levels
paralleled plasma cotinine levels. One can use steady-state plasma cotinine
levels to estimate daily systemic intake of nicotine.16 Based on population data
for the extent of conversion of nicotine to cotinine and for the clearance of
cotinine, the following equation computes nicotine intake from steady-state
cotinine levels: Dnic(mg) = Ccotcss(ng/ml) x 0.08, where Dnic is the daily dose
of nicotine, and Ccotcss is the steady state plasma cotinine concentration.16
In our subjects, plasma cotinine averaged about 15 ng/ml, indicating a daily
intake of 1.2 mg of nicotine. The comparison of the ETS-exposed group with the
non-ETS-exposed group once again revealed no difference in plasma cotinine
values--either at baseline or at any other time point--indicating that exposure
at work was the major contributor to plasma cotinine in the exposed group.
Urine nicotine tended to increase throughout the work day, but this change
was not significant, given the high variability observed.17,18 In addition,
urine cotinine increased nonsignificantly across the workshift. Urinary
excretion of cotinine was highly correlated with plasma cotinine value. Nicotine
and cotinine levels measured in our study were approximately one order of
magnitude lower than those observed in other studies of workers with GTS.19-21
Most of the previous studies, however, were performed in the United States and
India during the late 1970s or early 1980s, and some of the differences might
reflect an overall improvement of current working conditions. Another possible
explanation is the different farming practices or personal behaviors that might
occur among farmers from different countries.22,23 Finally, we studied
professional tobacco harvesters who were less likely to be exposed to excessive
amounts of nicotine than were inexperienced workers who were studied by other
investigators.
We attempted to estimate the external dose of exposure by applying paper pads
as a personal passive sampler. Paper pads have long been used as personal
passive samplers to monitor pesticide exposure of farmers.24 Nicotine is
absorbed through the skin; therefore, we decided to monitor nicotine exposure by
applying pads directly on the skin below clothing. The amount of nicotine
recovered from the pads was highly variable within the group and was not
correlated with any other absorption indicator. It is possible that different
behaviors of the study subjects might have occasionally contaminated the pads by
direct contact with tobacco leaves.
In this study we describe, for the first time, a complete profile of nicotine
and cotinine absorption and excretion in tobacco harvesters. However, there are
some limitations that merit consideration. Urine containers for after-shift
urine collection were given to subjects and kept in their houses until the
following day. Although we advised them to avoid exposing containers to tobacco
smoke, some contamination could have occurred. The diets of our subjects were
not standardized. Nicotine is present in some vegetables and beverages25;
therefore, some influence of the diets on the values observed cannot be excluded.
Nonetheless, the low values in women who did not harvest tobacco and values in
tobacco harvesters 3 mo following the end of the harvesting season provided
evidence that the\re was no significant contribution of nicotine from food.
In conclusion, the results of our study indicated that harvesting tobacco in
Italy exposed workers to a daily absorption of nicotine that is equivalent to
smoking 1 cigarette. Their cumulative nicotine exposure was equivalent to 180
cigarettes per season and 5,000 cigarettes during their working life. We find it
difficult to estimate the health effects of this nicotine absorption by
comparison to smokers because, unlike smokers, tobacco workers are not exposed
to tar, carbon monoxide, and other combustion products of smoked tobacco.26
Nicotine per se stimulates the sympathetic nervous system, increases heart rate
and blood pressure slightly, and may adversely affect lipid metabolism and
endothelial function. It is unclear whether these effects contribute
significantly to tobacco-related cardiovascular disease. Perhaps of more concern
for women who harvest tobacco is the possibility of nicotine toxicity during
pregnancy.27 Nicotine exposure in rodents during pregnancy causes impaired fetal
brain development28,29 and neonatal behavioral abnormalities.30 We do not know
if the low levels of nicotine found in our tobacco workers were toxic to their
cardiovascular and reproductive systems.
The authors thank Dr. Peyton Jacob for supervising the analytical chemistry
analyses and Silvia Wu and Lisa Yu for performing analytical chemistry
measurements.
This study was supported, in part, by the United States Public Health Service
Grants DA02277 and DA01696 awarded by the National Institute on Drug Abuse.
Submitted for publication April 17, 2000; revised; accepted for publication
January 15, 2001.
Requests for reprints should be sent to Alessandra d'Alessandro, M.D.,
Istituto di Medicina del Lavoro, Via E. dal Pozzo, Perugia 06122 Italy.
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ALESSANDRA D'ALESSANDRO Dipartimento di Medicina Clinica e
Sperimentale-Istituto di Medicina del Lavoro University di Perugia Perugia,
Italy
NEAL L. BENOWITZ
Division of Clinical Pharmacology and Experimental Therapeutics University of
California San Francisco, California
GIACOMO MUZI
Dipartimento di Medicina Clinica e Sperimentale-Istituto di Medicina del
Lavoro University di Perugia Perugia, Italy
MARK D. EISNER
Division of Occupational and Environmental Medicine and Division of Pulmonary
and Critical Care Medicine University of California San Francisco, California
SABRINA FILIBERTO
Dipartimento di Medicina Clinics e Sperimentale-Istituto di Medicina del
Lavoro Universita di Perugia Perugia, Italy
PAOLO FANTOZZI LUIGI MONTANARI
Dipartimento di Scienze e Tecnologie Alimentari e della Nutrizione Istituto
di Industrie Agrarie UniversitA di Perugia Perugia, Italy
GIUSEPPE ABBRITTI
Dipartimento di Medicina Clinica e Sperimentale-Istituto di Medicina del
Lavoro Universita di Perugia Perugia, Italy
Copyright HELDREF PUBLICATIONS May/Jun 2001
Publication date: 2001-05-01
© 2001, YellowBrix,
Inc.