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Biotransformation of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone in Lung Tissue from Mouse, Rat, Hamster, and Man

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Exposure to the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be an important etiological risk factor for lung cancer in tobacco users. The metabolism of NNK via carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), alpha-hydroxylation to form both DNA methylating and pyridyloxobutylating intermediates, and detoxification by pyridyl N-oxidation and glucuronide formation are well-characterized in laboratory animals but less so in man. The in vitro kinetics of 0.03-250 microM [5-(3)H]NNK metabolism were determined under identical experimental conditions using female A/J mouse, male Fischer 344 rat, female Syrian golden hamster, and human lung tissue explants in tissue culture. The concentration-dependent percentage contribution of the three major pathways of NNK metabolism (carbonyl reduction, alpha-hydroxylation, and N-oxidation) showed large interspecies variation. Quantitatively, in mouse, carbonyl reduction to NNAL increased steadily with an increasing substrate concentration (10-74% total NNK metabolism), while concurrent decreases occurred in end products of alpha-hydroxylation (60 to 18%) and N-oxidation (42 to 5%). In rat lung, there were no apparent concentration-dependent trends (NNAL, 42 +/- 4%; alpha-hydroxylation, 35 +/- 2%; and N-oxidation, 24 +/- 3%). In hamster lung, a clear concentration-dependent increase in the contribution of NNAL to total NNK metabolism (from 47 to 87%) was paralleled by a steady decline in end products of alpha-hydroxylation (31 to 11%) and N-oxidation (22 to 2%). Human lung metabolism showed no concentration-dependent tendencies (NNAL, 89 +/- 1%; alpha-hydroxylation, 8.8 +/- 1.1%; and N-oxidation, 2.1 +/- 0.3%). The major alpha-hydroxylation product in human lung was 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), thus supporting the potential pyridyloxobutylation of lung DNA. Metabolism to 4-(3-pyridyl)-4-oxobutanoic acid (keto acid), which could result in lung DNA methylation, was only sporadically seen in human lung but present to a far greater extent in rodent lung. No evidence for glucuronidation was found in any species. Generally, the rate of formation of all NNK metabolites showed two different enzyme kinetics, resulting in large differences between apparent K(m) and V(max) values in the low (up to 2.8 microM) and high substrate concentration ranges. The metabolism of NNK by alpha-hydroxylation is considerably lower in human lung as compared to that observed in rodent species, suggesting that extrapolation of in vitro rodent data to man may result in invalid conclusions about the capacity of the human lung to activate NNK under realistic conditions of NNK exposure expected to occur in man.
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ORIGINAL ARTICLE
E. Richter áS. Ro
Èsler áG. Scherer áJ. G. Gostomzyk
A. Gru
Èbl áU. Kra
Èmer áH. Behrendt
Haemoglobin adducts from aromatic amines in children in relation
to area of residence and exposure to environmental tobacco smoke
Received: 23 August 2000 / Accepted: 12 April 2001
Abstract Objective: The in¯uence of area of residence
on haemoglobin (Hb) adducts of 4-aminobiphenyl
(4-ABP), o-, m-, p-toluidine and o-anisidine was inves-
tigated in children from three dierent-sized Bavarian
cities ± Munich, Augsburg and Eichsta
Ètt, with 1,300,000,
250,000 and 13,000 inhabitants, respectively ± and was
compared with that of exposure to environmental
tobacco smoke (ETS). Methods: Blood samples from
Munich (n34) and Eichsta
Ètt (n64) were from chil-
dren attending the Paediatric Clinic of the Technical
University of Munich (TUM) or a practice in Eichsta
Ètt,
respectively. Blood samples (n126) together with
urine samples (n88) were collected from Augsburg
children during school medical examination. Personal
data including possible sources of ETS exposure were
obtained at the interview. Hb adduct levels were ana-
lysed by a gas chromatographic method, using mass
spectrometry with selected-ion monitoring. Urinary
cotinine was determined by radioimmunoassay. Results:
4-ABP Hb adduct levels in children from Munich were
1.5 and 1.2 times higher than those in children from
Eichsta
Ètt and Augsburg (P<0.001). Children from
Munich also had signi®cantly higher Hb adduct levels
of monocyclic aromatic amines than did children from
Eichsta
Ètt and, except for o-toluidine, children
from Augsburg (P<0.005). Compared with children
from Eichsta
Ètt, children from Augsburg had higher Hb
adduct levels of 4-ABP, o- and m-toluidine (P<0.01) but
not p-toluidine and o-anisidine. In a multivariate anal-
ysis, gender, age and body mass index had no consistent
in¯uence on Hb adducts. ETS exposure resulted in a
slight, nonsigni®cant increase in 4-ABP Hb adduct
levels. In contrast, adduct levels from monocyclic aroma-
tic amines were consistently decreased in ETS-exposed
children (signi®cant for o- and m-toluidine, P<0.05).
Conclusions: Hb adducts from aromatic amines in chil-
dren were strongly in¯uenced by site of residence,
whereas ETS exposure did not signi®cantly increase the
adduct levels.
Key words Haemoglobin adducts áAromatic
amines áChildren áRegional dierence áPassive
smoking
Introduction
A weak association between exposure to environmental
tobacco smoke (ETS) and lung cancer has been repeat-
edly found in epidemiological studies (Dockery and
Trichopoulos 1997). In a meta-analysis of 30 studies the
relative risk for lung cancer in nonsmoking women ex-
posed to ETS was estimated to be 1.19 (90% con®dence
interval 1.04±1.35; US EPA 1992). However, because of
Int Arch Occup Environ Health (2001) 74: 421±428 ÓSpringer-Verlag 2001
E. Richter (&)áS. Ro
Èsler
Walther Straub-Institut fu
Èr Pharmakologie und
Toxikologie, Ludwig-Maximilians-Universita
ÈtMu
Ènchen,
Nussbaumstrasse 26, 80336 Munich, Germany
e-mail: franky.richter@lrz.uni-muenchen.de
Fax: +49-89-51607207
G. Scherer
Analytisch-biologisches Forschungslabor, Munich, Germany
J. G. Gostomzyk
Gesundheitsamt der Stadt Augsburg, Augsburg, Germany
A. Gru
Èbl
Kinderklinik und Poliklinik der Technischen Universita
Èt
Mu
Ènchen, Munich, Germany
U. Kra
Èmer
Medizinisches Institut fu
Èr Umwelthygiene,
Abteilung Epidemiologie, Du
Èsseldorf, Germany
U. Kra
Èmer
Klinik und Poliklinik fu
Èr Dermatologie und Allergologie
am Biederstein, Technische Universita
ÈtMu
Ènchen, Munich,
Germany
H. Behrendt
Klinische Kooperationsgruppe Umweltdermatologie
und Allergologie GSF/Technische Universita
ÈtMu
Ènchen,
Munich, Germany
potential confounding the validity of relative risks well
below 2 is questionable (Taubes 1995; U
Èberla 1998). A
recent multicentre case-control study in Europe, one of
the largest and most exhaustive examinations, failed to
show a statistically signi®cant increase in risk by spousal
and/or workplace exposure to ETS (combined odds
ratio for ever-exposure 1.14: 95% con®dence inter-
val 0.88±1.47). In contrast, ETS exposure during
childhood resulted in a signi®cantly decreased risk of
lung cancer (odds ratio for ever-exposure 0.78: 95%
con®dence interval 0.64±0.96; Boetta et al. 1998). In
order to improve the weak evidence of an ETS-related
risk, biomarker studies on dose and eect of ETS ex-
posure were performed (Scherer and Richter 1997). A
signi®cant, higher uptake of carcinogens from ETS than
from other environmental sources would substantially
contribute to the plausibility of a true increase in cancer
risk by ETS (Blot and McLaughlin 1998). One of the
most prominent examples of such an increased exposure
is the study by Hammond et al. (1993) showing a sig-
ni®cant correlation between Hb adduct levels from the
human carcinogen 4-aminobiphenyl (4-ABP) and expo-
sure to ETS in nonsmoking pregnant women. However,
in a more recent study we were not able to con®rm this
®nding. Hb adducts from neither 4-ABP nor from other
aromatic amines or from tobacco-speci®c nitrosamines
showed a relationship to ETS exposure in pregnant
women (Branner et al. 1998). Therefore, in our view the
major source of these adducts in nonsmokers is still
unknown. In previous studies, we found evidence that
aromatic amine Hb adduct levels are lower in people
living in rural areas compared with urban environments
(Falter et al. 1994).
The objective of this study was to elucidate the in-
¯uence of place of living and ETS exposure on Hb ad-
duct levels of aromatic amines in nonsmokers. For this
purpose Hb adduct levels were measured in children
living in three cities which largely dier in their sizes.
Subjects and methods
Subjects
The study was performed with children living in three cities in
Southern Germany: Munich, the capital of Bavaria with 1.3 million
inhabitants; Augsburg, 30 miles west of Munich, with 250,000 in-
habitants; Eichsta
Ètt, 45 miles north of Munich, with 13,000 in-
habitants. Only the erythrocyte fractions of blood samples drawn
for other purposes were used in the present study. Written consent
from the children's parents or guardians was obtained for each
sample.
In Spring 1996, all 2,444 6 to 7-year-old children from Augs-
burg starting school in fall 1996 were invited to take part in an
allergological and dermatological investigation, called MIRIAM
(Multicentric International Study for Risk Assessment of Indoor
and Outdoor Air on Allergy and Neurodermitis Morbidity). The
purpose of the MIRIAM study was to evaluate the impact of
outdoor and indoor air pollution on sensitizations and allergies in
children. The investigation was combined with the school entrance
medical examination which is compulsory for all school beginners,
and 1,669 (69%) children agreed to participate. During the ®rst
6 weeks of the study, the erythrocyte fractions of blood samples
from 126 randomly selected children were obtained for determi-
nation of aromatic amine Hb adducts. Spot urine samples were
available from 88 of the 126 children. Blood samples from the
Munich subjects were obtained during fall 1996 from 34 children
attending the Paediatric Clinic of the Technical University of
Munich. All children were diagnosed for general clinical symptoms
and for atopic allergy according to the criteria of the ongoing GINI
(German Infant Nutritional Intervention) programme. Brie¯y, the
diagnosis of atopic allergy was based on clinical symptoms, positive
skin ``prick'' test and the negative radio allergen sorbent test
(RAST). For Eichsta
Ètt, blood samples were provided by the pae-
diatrician Dr. K. Wenk from 65 children consulting the practice for
routine check-ups or various diseases during fall 1996.
For all children from Munich and Eichsta
Ètt a single page
questionnaire was completed by the investigators, addressing
questions of general health, life-style, and exposure to ETS. From
the Augsburg children a validated questionnaire with 70 questions
on air-way diseases, allergies and potentially confounding factors
was obtained. The sociodemographic data are summarized in
Table 1. Based on the subjects' living conditions, exposure to ETS
was strati®ed into three categories: A, no ETS exposure; B, expo-
sure by household members other than the mother; C, exposure via
maternal smoking.
A blood sample (10 ml) was collected in EDTA-treated Vacu-
tainers. A spot urine sample was obtained from 88 of the 126
children from Augsburg and stored at ±20 °C. Plasma was sepa-
rated from blood cells by centrifugation and the blood cells were
washed three times with 8 ml of saline. Blood samples from
Augsburg and Eichsta
Ètt were processed immediately and stored at
±20 °C prior to shipment to our laboratory on dry ice. Samples
from Munich were kept at 4 °C and transported on ice within 6 h
to our laboratory for further processing.
Analytical methods
Hb adducts
Aromatic amine Hb adducts were determined as previously de-
scribed, with some minor modi®cations (Kutzer et al. 1997).
Brie¯y, Hb solutions obtained after centrifugation of lysed red
Table 1 Sociodemographic data of the study population. ETS
score gives numbers of children with Ano ETS exposure, Bex-
posure by household members other than the mother, Cexposure
to maternal smoking; mean  SD (n; min./max.)
Characteristic Munich Augsburg Eichsta
Ètt
Total number 34 126 64
Size of city 1,300,000 250,000 13,000
Gender
(male/female)
25/9 64/59 36/28
Age (years) 9.3  2.3 6.3  0.3
b
8.2  3.6
c
(34; 5/15) (118; 5.9/6.9) (63; 0.25/14)
BMI (kg/m) 17.3  2.9 16.0  1.9
a
16.7  2.7
(27; 11.2/23.1) (118; 10.8/22.7) (63; 12.3/23.3)
ETS exposure 3.28  1.94 6.50  6.41 1.95  0.98
(h/d) (17; 0.25/8.0) (46; 1.0/24.0) (16; 0.25/3.5)
ETS score
A 15 (45%) 65 (52%) 39 (61%)
B 5 (15%) 31 (25%) 16 (25%)
C 13 (39%) 27 (22%) 9 (14%)
a
Signi®cantly dierent from Munich; P<0.05
b
Signi®cantly dierent from Munich; P<0.0001
c
Signi®cantly dierent from Augsburg; P<0.001
422
blood cells were dialysed against 20-times the volume of deionized
water for 2 days with three changes of the water. Hb content was
determined by Drabkin's assay (Sigma, Deisenhofen, Germany).
The samples were divided into two equal parts. After mild base-
catalysed hydrolysis 78 pg D
4
-p-toluidine (a gift from Prof.
Sabbioni, Walther Straub Institute, Munich, Germany) and 81 pg
D
9
-4-ABP (IC Chemikalien, Munich, Germany) were added as
internal standards. Extraction, clean-up and concentration were
performed by a one-step procedure using C
18
cartridges (Varian,
Darmstadt, Germany). The cartridges were eluted in three steps
with 1.5, 1.0 and 0.75 ml CHCl
3
, and the combined CHCl
3
extracts
were concentrated in a vacuum centrifuge. The aromatic amines
were derivatized with penta¯uoropropionic anhydride and ana-
lysed by capillary gas chromatography-mass spectrometry with
negative chemical ionization and selected-ion monitoring. The
analytical limit of detection was 0.5±1 pg adduct/g Hb using a 5-ml
aliquot of blood. All samples were analysed in duplicate. Two
blank water samples were analysed each day to control for back-
ground contamination.
Urinary cotinine
Cotinine in urine was determined by a radioimmunoassay ac-
cording to the method of Langone et al. (1973), with modi®cations
by Haley et al. (1983). The limit of detection was 1 ng/ml. Creati-
nine in urine was determined by the Jae
Âmethod using a com-
mercial test kit (Merck, Darmstadt, Germany). Results on cotinine
are given as the cotinine (lg) to creatinine (g) ratio (CCR).
Statistical analysis
When not stated otherwise, results are given as arithmetic means
and standard deviations (SDs). All haemoglobin adduct concen-
trations were normally distributed after logarithmic transformation
(base 10). Therefore, all further statistical tests were done after this
transformation. Comparison of group means was performed by
Student's t-test. The in¯uence of ETS adjusted for place and socio-
demographic parameters, as well as the in¯uence of place adjusted
for ETS and sociodemographic parameters, was determined by
linear regression. The resulting parameter estimates (b) were
transformed: f(b) 10
b
a, where a can be interpreted as an ad-
justed quotient of geometric means (mean ratio, MR), indicating
the change in geometric means when the independent variable
changes by one unit. For binary variables (ETS yes or no, for
instance) MR is the adjusted change in geometric means when the
factor is present, compared with the case when the factor is not
present. If the respective 95% con®dence interval (95% CI) does
not include 1 (= no change) the result is judged signi®cant.
Results
Hb adducts of 4-ABP, o-, m-, p-toluidine, and o-anisi-
dine were detectable in all blood samples. Duplicate
analyses revealed a mean coecient of variation of 4%.
In all but one sample the adduct levels of 3-aminobi-
phenyl were below the limit of detection (1 pg/g Hb).
In children from Eichsta
Ètt, four Hb adduct levels each
of m-toluidine and p-toluidine (<176 and<79 pg/g Hb,
respectively) were below the threefold interquartile dif-
ference from the median values and were, therefore,
regarded as outliers, and were eliminated from the sta-
tistical analyses.
All aromatic amine Hb adduct levels were highest in
children from Munich, intermediate in children from
Augsburg and lowest in children from Eichsta
Ètt (Fig. 1,
Table 2). The dierences in biomarkers between children
from Munich and Eichsta
Ètt were statistically signi®cant
for 4-ABP (1.5-fold; P<0.001), o-andp-toluidine (1.3
to 1.6-fold; P<0.001) as well as o-anisidine (1.1-fold;
P0.004). With the exception of o-toluidine (1.1-fold;
P0.058) adduct levels were also statistically higher in
children from Munich than in children from Augsburg
(1.2±1.3 times; P<0.001). The Hb adduct levels in
children from Augsburg were signi®cantly higher than in
children from Eichsta
Ètt for 4-ABP (1.3 times;
P0.009), o-toluidine (1.2 times; P0.003) and
m-toluidine (1.3 times; P0.002) but not p-toluidine
and o-anisidine.
With only two exceptions, all dierences between
children from Eichsta
Ètt and Munich were nearly iden-
tical and remained signi®cant when children from non-
smoking (ETS no) and smoking homes (ETS yes) were
Fig. 1 Regional dierences in aromatic amine Hb adduct levels.
Boxes cover 25th and 75th percentiles with median levels indicated
as horizontal lines. Outer horizontal lines indicate 5th and 95th
percentiles.
a
Signi®cantly dierent from Munich, P<0.01,
b
signi-
®cantly dierent from Munich, P<0.001,
c
signi®cantly dierent
from Augsburg, P<0.01
423
compared separately (Table 2). For o-toluidine and
o-anisidine the dierences in children from nonsmoking
households were weaker and not more signi®cant. Sim-
ilar results were obtained for the dierences between
children from Augsburg and Munich, which remained
signi®cant after separate analysis according to the ETS
exposure with the exception of o-anisidine. For the
comparison of children from Augsburg and Eichsta
Ètt,
the signi®cant dierences for m-toluidine remained,
whereas the dierences for 4-ABP were lost in both
subgroups and for o-toluidine and o-anisidine only in
children from nonsmoking homes.
In all three towns children from nonsmoking homes
had nonsigni®cantly lower 4-ABP adduct concentrations
than children from smoking homes (Table 2). In con-
trast, monocyclic aromatic amine adduct levels were
always higher in children from nonsmoking homes in
Augsburg and Eichsta
Ètt, reaching signi®cance for o- and
m-toluidine in children from Eichsta
Ètt (1.8- and 1.3-fold,
P<0.01). In children from Munich no in¯uence on
monocyclic aromatic amine adduct levels by smoking
status of the households was obvious.
The validity of self-reported ETS exposure was con-
®rmed by determination of urinary cotinine in children
from Augsburg. Children from smoking homes (n38,
48.1  40.7 lg cotinine/g creatinine) had a more than
3-times higher CCR than children from nonsmoking
homes (n50, 13.9  12.7 lg/g, P<0.001). In con-
trast, Hb adduct levels were not signi®cantly dierent
between exposed and nonexposed children. Whereas
4-ABP adducts were slightly higher in children from
smoking than in children from nonsmoking homes
(29.3  21.7 versus 23.3  14.4 pg/g, P0.058), ad-
ducts from monocyclic amines were consistently de-
creased by about 10%±20% (dierences not signi®cant,
P>0.1). As shown in Fig. 2, children exposed to ETS
via maternal smoking (n19) had a signi®cantly higher
CCR (62.4  49.5 lg/g) than children exposed to ETS
by household members other than the mother (n19,
33.8  23.1 lg/g, P0.031), and in both exposed
groups the CCR was signi®cantly higher than in children
from nonsmoking households (P<0.001). In contrast,
4-ABP Hb adduct levels were not increased in children
with smoking mothers compared with children exposed
to ETS by other household members (29.1 24.0
versus 29.6  19.9 pg/g).
Table 2 Regional dierences in
haemoglobin adducts of
aromatic amines (pg g
)1
Hb) in
children; means  SD (n)
Statistical dierences (by
Student's t-test: ) are
determined between towns in
corresponding groups (all, ETS
yes, ETS no) and within a town
between ETS yes or no
Hb adduct Munich Augsburg Eichsta
Ètt
4-ABP All 30.7  6.1 (33) 26.6  17.5 (123)
b
20.7  11.8 (64)
b,c
ETS no 30.1  7.0 (15) 25.0  14.8 (65)
b
19.0  7.9 (39)
b
ETS yes 31.2  5.5 (18) 28.5  20.0 (58)
a
23.4  15.9 (25)
a
o-Toluidine All 632  206 (33) 598  298 (123) 487  295 (64)
b,c
ETS no 620  206 (15) 621  327 (65) 558  328 (39)
f
ETS yes 642  212 (18) 574  262 (58) 376  191 (25)
b,d
m-Toluidine All 1,384  259 (33) 1,115  416 (123)
b
935  408 (60)
b,c
ETS no 1,410  287 (15) 1,143  418 (65)
b
1,014  439 (37)
b,e
ETS yes 1,363  240 (18) 1,084  422 (58)
b
809  321 (23)
b,c
p-Toluidine All 1,254  279 (33) 991  485 (123)
b
866  408 (60)
b
ETS no 1,229  258 (15) 1,018  498 (65)
b
906  367 (38)
b
ETS yes 1,275  300 (18) 944  468 (58)
b
795  302 (22)
b
o-Anisidine All 284  73 (33) 242  129 (123)
b
254  179 (64)
a
ETS no 275  81 (15) 256  144 (65) 274  200 (39)
ETS yes 292  66 (18) 225  111 (58)
b
222  138 (25)
a
a
Signi®cantly dierent from Munich; P<0.01
b
Signi®cantly dierent from Munich; P<0.001
c
Signi®cantly dierent from Augsburg; P<0.01
d
Signi®cantly dierent from Augsburg; P<0.001
e
Signi®cantly dierent from children exposed to ETS; P<0.05
f
Signi®cantly dierent from children exposed to ETS; P<0.01
Fig. 2 In¯uence of ETS exposure on 4-ABP Hb adduct levels and
urinary cotinine excretion in 88 children from Augsburg. ETS
exposure groups: Ano ETS exposure, Bexposure by household
members other than the mother, Cexposure to maternal smoking.
Boxes cover 25th and 75th percentiles, with median levels indicated
by horizontal lines. Outer horizontal lines indicate 5th and 95th
percentiles.
a
A<B and A<C, P<0.001;
b
B<C, P0.031
424
The results of linear regression analysis on the in¯u-
ence of gender, age, body mass index (BMI), ETS ex-
posure and place of residence on Hb adduct levels are
given in Table 3. Whereas BMI did not show any sig-
ni®cant eect, the levels of Hb adducts of the toluidine
isomers tended to be lower in boys than in girls. Age was
without eect on Hb adducts except for a slight decrease
in m-toluidine adduct levels with increasing age. Chil-
dren living in smoking homes exhibited a small, non-
signi®cant increase in 4-ABP adduct levels and a small
but signi®cant decrease in adduct levels from toluidines.
The linear regression analysis con®rmed the regional
dierences in Hb adducts from aromatic amines. Using
children from Eichsta
Ètt as a reference group, we found
that MRs were consistently greater than 1.0, reaching
signi®cance for all aromatic amines in children from
Munich and for o-toluidine and m-toluidine in children
from Augsburg.
Discussion
In this study internal exposure to aromatic amines has
been studied in children by an established analytical
method (Tannenbaum and Skipper 1994) slightly mod-
i®ed by us (Kutzer et al. 1997), and acknowledged after
further minor modi®cations by the German Commission
for the Investigation of Health Hazards of Chemical
Compounds in the Work Area (Lewalter and Gries
(2000). Adducts from 4-ABP, o-, m-andp-toluidine
isomers, as well as from o-anisidine, were detected in all
samples. With the exception of m-toluidine, all other
amines have been classi®ed as carcinogens (Sabbioni and
Richter 1999). The adduct levels of 4-ABP were in the
same concentration range as the levels reported in chil-
dren from New York (Tang et al. 1999) and in adult
nonsmokers (Bartsch et al. 1990; Bryant et al. 1987;
Falter et al. 1994; Hammond et al. 1993, Pinorini-Godly
and Myers 1996; Rielmann et al. 1995). Adducts from
monocyclic aromatic amines have been determined for
the ®rst time in children. The concentrations of tolui-
dines are 2±3 times higher than reported for adults
(Bryant et al. 1988; Falter et al. 1994). A possible ex-
planation for this discrepancy could be that we used a
more appropriate internal standard, D
4
-p-toluidine,
compared with D
5
-aniline in the earlier studies. Using
this internal standard we realized that in our original
analytical method (Kutzer et al. 1997) the elution of the
toluidines from the C
18
cartridges was incomplete. A
better recovery together with a more precise calculation
based on the new internal standard could have led to the
higher values in this study. The presence of o-anisidine
Hb adducts in children is not surprising. Adducts from
this amine have been detected previously in adults from
Germany (Branner et al. 1998, Falter et al. 1994). The
origin of this adduct is unknown. o-Nitroanisole, a
possible precursor, was released into the environment in
the course of an accident in a German chemical plant
(Hauthal 1993).
Table 3 Result of linear regression; adjusted mean ratios (MR) and 95% con®dence intervals (95% CI) with Pvalues
Characteristic 4-ABP (n= 207) o-Toluidine (n= 207) m-Toluidine (n= 204) p-Toluidine (n= 203) o-Anisidine (n= 207)
MR 95% CI P< MR 95% CI P< MR 95% CI P< MR 95%CI P< MR 95%CI P<
Gender
Female 1.00 1.00 1.00 1.00 1.00
Male 0.99 0.86±1.15 0.94 0.82±1.08 0.91 0.82±1.01 0.10 0.88 0.78±0.99 0.05 1.03 0.90±1.18
Age (per year) 0.97 0.94±1.01 1.00 0.96±1.04 0.97 0.94±1.00 0.05 0.98 0.95±1.01 1.01 0.97±104
BMI (per unit) 1.02 0.99±1.06 1.01 0.98±1.05 0.99 0.97±1.02 0.98 0.95±1.01 0.98 0.95±1.01
ETS
No exposure 1.00 1.00 1.00 1.00 1.00
Smoking home 1.10 0.95±1.27 0.86 0.74±0.98 0.05 0.88 0.80±0.98 0.05 0.90 0.79±1.01 0.01 0.91 0.79±1.04
City
Eichsta
Ètt 1.00 1.00 1.00 1.00 1.00
Augsburg 1.18 0.99±1.40 0.10 1.27 1.08±1.50 0.01 1.14 1.00±1.29 0.05 1.01 0.87±1.17 0.99 0.85±1.17
Munich 1.68 1.32±2.16 0.01 1.40 1.11-1.77 0.01 1.68 1.41±1.99 0.01 1.55 1.26±1.92 0.01 1.26 1.02±1.59 0.05
425
Our ®ndings show that children living in Eichsta
Ètt, a
small town with a largely rural environment, had con-
siderably lower aromatic amine Hb adduct levels than
children from larger cities such as Augsburg and
Munich. Tobacco smoke is certainly not the only source,
and probably not the most important one, because there
was no signi®cant increase in these adducts with expo-
sure of the children to ETS (Tables 2 and 3). At present,
little is known about sources other than tobacco smoke
for the human uptake of aromatic amines or their cor-
responding nitro-compounds, e.g. 4-nitrobiphenyl, as
precursors (Bryant et al. 1987). Air pollution associated
with trac density could be a source of aromatic amine
Hb adducts which may arise from the uptake of nitro-
aromatics present in diesel exhaust (Bryant et al. 1987;
Ries 1992). Kerosene heaters, e.g. open-type oil-burning
heaters (Tokiwa and Ohnishi 1986), were used in only a
few households of our study subjects and were not as-
sociated with increased Hb adduct levels. Other possible
sources for these amines or their nitroaromatic precur-
sors, including food (Bryant et al. 1987; Neurath et al.
1977; Richter et al. 2000; Vitzthum et al. 1975) and
drinking water (Djozan and Faraj-Zadeh 1995; Fattore
et al. 1998; Mu
Èller et al. 1997; Neurath et al. 1977) or
azo-dyes (Bryant et al. 1987; Oh et al. 1997; Platzek
et al. 1999) are not expected to dier signi®cantly
between the three cities.
Selection bias is not likely to explain these results.
Age and BMI had no in¯uence on Hb adduct levels. The
tendency for lower adduct levels in boys compared with
girls would have rather diminished the dierences be-
tween Munich (74% boys) and the other cities (52%
boys in Augsburg and 56% boys in Eichsta
Ètt). To the
best of our knowledge, these variables have not been
addressed in other studies on Hb adducts from aromatic
amines.
In a smaller study with 51 preschool children from
New York, Tang et al. (1999) measured 4-ABP Hb
adduct levels of 32  2 pg/g Hb, similar to our values
for children from Munich (31  6 pg/g). In a subgroup
of ten children living in nonsmoking households in New
York the adduct levels were 31% lower than in 41 New
York children living in smoking homes (Tang et al.
1999). This dierence is higher than the dierence of
10% which we observed after adjustment in our
207 children. However, the dierence in the New
York children, 47 Hispanic and four African-American
subjects, reached signi®cance (P<0.05) only after
adjustment for ethnicity. Ethnicity did not play a role in
our study since all children were of Caucasian origin.
One reason for the higher impact of ETS exposure on
4-ABP Hb adduct levels in the study from Tang et al.
(1999) compared with our study could be the much
higher extent of ETS exposure in the New York
children. Plasma cotinine levels in children living in
smoker households were 2.87  5.61 ng/ml, compared
with 0.264  0.596 ng/ml in children from nonsmoking
households, an 11-fold dierence. In our study, the
Augsburg children from smoking homes had only 3.5-
times higher urinary cotinine levels than children from
nonsmoking homes. However, when we compared the
23 children with the highest CCR with the 23 children
with the lowest CCR, thereby obtaining a similar 11-fold
dierence in ETS exposure (65.0 8.1 versus 6.1
0.5 lg/g; P<0.001) compared with the study from
Tang et al. (1999), no dierence in 4-ABP Hb adduct
levels (23.9  1.9 versus 24.3  3.5 pg/g; P>0.1)
was seen. Interestingly, in this subgroup, Hb adduct
levels of all three toluidine isomers were signi®cantly
lower in the highly exposed children from smoking
homes than in the children from nonsmoking homes
(o-toluidine: 574  58 versus 757  66 pg/g, P0.042;
m-toluidine: 1,119  83 versus 1,384  87 pg/g, P
0.033; p-toluidine: 979  109 versus 1,304  107 pg/g,
P0.039). The dierence for o-anisidine (242  26
versus 328  36 pg/g, P0.057) did not reach signi®-
cance.
The lack of a signi®cant eect of ETS exposure on
4-ABP Hb adduct levels is in accordance with our
previous study in pregnant women (Branner et al.
1998). More recently, Grimmer et al. (2000) did not
®nd a dierence between urinary 4-ABP levels in
smokers, nonsmokers and passive smokers. In the
present study, an ETS-related reduction, rather than an
increase, in toluidine Hb adduct levels has been ob-
served, con®rming the results of Branner et al. (1998).
We still have no explanation for this paradoxical result.
In smokers, these adducts are consistently elevated
(Branner et al. 1998; Ronco et al. 1990) in accordance
with the well-documented occurrence of toluidines in
tobacco smoke (Grimmer and Schneider, 1995; Luceri
et al. 1993; Patrianakos and Homann 1979). How-
ever, the dierences in Hb adducts from toluidines
between smokers and nonsmokers were much less than
those from 4-ABP. Only a small nonsigni®cant dier-
ence was also reported for urinary o-toluidine in
smokers and nonsmokers (El-Bayoumy et al. 1986). It
could be speculated that dierent dietary habits in
smoking versus nonsmoking households may be re-
sponsible for the increased toluidine Hb adduct levels
in children from nonsmoking homes. Dietary items
themselves could be a source of these amines (Neurath
et al. 1977; Vitzthum et al. 1975). Laboratory rats were
found to have higher Hb adduct levels of toluidines and
4-ABP than active smokers (Green et al. 1984; Richter
et al. 2000; Haussmann et al. 1998). In rats, the adducts
originated most probably from the food pellets (Richter
et al. 2000). Enzyme induction by high intake of vita-
mins (Lutz et al. 1998; Paolini et al. 1999), leading to
more extensive metabolic activation of aromatic amines
via N-hydroxylation, could be another possibility if one
assumes that in nonsmoking homes healthier and more
vitamin-rich diets are consumed (Subar et al. 1990;
Matanoski et al. 1995). In the case of 4-ABP, a diet-
related increase in Hb adduct levels may be masked by
a higher ETS-related exposure to this carcinogen.
In summary, the results highlight the importance
of place of residence for the exposure of children to
426
carcinogenic aromatic amines, and suggest that the
contribution from passive smoke is comparably
small.
Acknowledgements The authors thank Dr. K. Wenk for collecting
blood samples and the questionnaire data of the children from
Eichsta
Ètt. This work was supported by a grant from VERUM,
Stiftung fu
Èr Verhalten und Umwelt.
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... [18][19][20][21][22][23] It is thought that NNK carcinogenicity is largely manifested via the NNAL formation pathway, with 14-100% of the NNK dose metabolized to NNAL. 15,[24][25][26][27][28] By measuring NNK in mainstream smoke vs urinary NNAL in smokers, it was estimated that 39-100% of NNK was converted to NNAL systemically in smokers. 26 NNK exposure in smokeless tobacco users was measured in saliva and it was estimated that 14-17% of NNK was converted to NNAL within the oral cavity. ...
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Tobacoo-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen present in tobacco products and tobacco smoke. The carcinogenic mechanism of NNK is involved in the DNA damage induced by the active electrophiles generated from the metabolic activation of NNK catalyzed by cytochrome P450. It is significant to establish an in vitro metabolism model of NNK and the quantitation method for the investigation of tabacco carcinogens motabolites. In this work, the NNK metabolite of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) was quantitatively analyzed by high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS). Selective reaction monitoring (SRM) was employed for determining HPB and the internal standard HPB simultaneously. The method shows good linearity within 0.2-400 nmol/L with the correlation coefficient (R2) 0.9999. The limit of detection (LOD) and the limit of quantitation (LOQ) are 0.025 nmol/L (S/N=3) and 0.05 nmol/L (S/N=10), respectively. The inter-day and intra-day accuracy range from 96.6% to 101.8%, and the recovery is 98.1%-102.6%. The in vitro metabolism model of NNK can be used to elucidate the molecular mechanism of the metabolic activation of tobacco-specific nitrosamine. This work layed a foundation for the quantification of biomarkers for tobacco carcinogenesis. ©, 2014, Journal of Chinese Mass Spectrometry Society. All right reserved.
Article
Among the most potent carcinogens in tobacco are the tobacco-specific nitrosamines (TSNAs), with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) the most potent as well as one of the most abundant. NNK is extensively metabolized to the equally carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Of the two NNAL enantiomers, (S)-NNAL appears to be preferentially glucuronidated and excreted in humans, but also exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its (R)- counterpart. Due to the differential carcinogenic potential of the NNAL enantiomers, it is increasingly important to know which UGT enzyme targets the specific NNAL enantiomers for glucuronidation. To examine this, a chiral separation method was developed to isolate entiomerically pure (S)- and (R)-NNAL. Comparison of NNAL glucuronides (NNAL-Glucs) formed in reactions of UGT2B7-, UGT2B17-, UGT1A9-, and UGT2B10-over-expressing cell microsomes with pure NNAL enantiomers showed large differences in kinetics for (S)- versus (R)-NNAL, indicating varying levels of enantiomeric preference for each enzyme. UGT2B17 preferentially formed (R)-NNAL-O-Gluc and UGT2B7 preferentially formed (S)-NNAL-O-Gluc. When human liver microsomes (HLM) were independently incubated with each NNAL enantiomer, the ratio of (R)-NNAL-O-Gluc to (S)-NNAL-O-Gluc formation in HLM from subjects exhibiting the homozygous deletion UGT2B17 (*2/*2) genotype was significantly lower (p=0.012) than HLM from wild-type (*1/*1) subjects. There was a significant trend (p=0.015) towards decreased (R)-NNAL-O-Gluc:(S)-NNAL-O-Gluc ratio with increasing numbers of the UGT2B17*2 deletion allele. These data demonstrate that variations in the expression or activity of specific UGTs may affect the clearance of specific NNAL enantiomers known to induce tobacco-related cancers.
Article
A hydrophilic-interaction liquid chromatography–tandem mass spectrometry (HILIC–MS–MS) method was developed for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites in mouse liver and lung. The limits of detection of all analytes were in the range 0.017–0.057 ng mL−1, and recovery ranged from 88.4–119.8 % with intra and inter-day precision in the range 0.89–6.03 % and 1.01–6.97 %, respectively. This simple and accurate method was used to evaluate the effect of chronic alcohol consumption on NNK bioactivation in mouse tissue. Time-course curves for NNK and its metabolites were generated, and the areas under the curves (AUCs) were compared. It was found that target tissues of NNK carcinogenesis in C57BL/6 mice contained high levels of α-hydroxylation metabolites of NNK and its carbonyl reduction metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The most pronounced effect of alcohol was to enhance α-hydroxylation of NNK in mouse lung and liver, which suggests that chronic alcohol consumption may increase the risk of carcinogenicity associated with NNK in mice. Figure ᅟ
Chapter
Drug-metabolizing enzymes (DMEs) are primarily expressed in the liver but their role in the extrahepatic tissues such as gastrointestinal tract (GIT), pulmonary, excretory, nervous, cardiovascular system, and skin cannot be neglected. Generally, the expression of DMEs in extrahepatic tissues is quantitatively lower than that in the liver, but there are a few enzymes such as CYP1A1, CYP1B1, CYP2F1, and CYP2U1 that are more abundant in extrahepatic organs. As many extrahepatic organs are portals for administered drugs, DMEs expressed in these organs can be responsible for significant metabolism, leading to first-pass effects and lower bioavailability. Extrahepatic DMEs are also involved in bioactivation of prodrugs and formation of reactive metabolites that may interact with cellular components, resulting in organ-specific toxicity. Activity and expression of extrahepatic DMEs is often altered by coadministered drugs, leading to drug–drug interactions. Expression of DMEs in living beings affected by a host of environmental and genetic factors such as genetic polymorphism, age, gender, pathophysiological conditions, inborn errors in metabolism, food habits, and environmental pollutants, contributing to varied drug effects and idiosyncratic toxicities.
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A method for the determination of aromatic amines in mainstream smoke of mechanically smoked cigarettes has been developed. The fast reaction of aromatic amines with other smoke constituents formed during the combustion process can be significantly reduced by the addition of an excess of p-toluidine to the acidic collecting solution. From this point the p-toluidine as intercept reactant immediately stabilizes the originally formed amines and results in high recovery rates. The method allows the gas chromatographic determination of aniline, o- and m-toluidine, 1- and 2-aminonaphthalene, 2- and 4-aminobiphenyl, 1-, 2- and 4-aminofluorene, 3-aminofluoranthene, 1-aminopyrene and 6-aminochrysene in the mainstream smoke of a single cigarette. d
Article
The exposure of non-smokers to the tobacco-specific N-nitrosamine 4-(N- methylnitrosamino)-1-3-pyridyl)-1-butanone (NNK), a rodent lung carcinogen, was determined in the air of various indoor environments as well as by biomonitoring of nonsmokers exposed to environmental tobacco smoke (ETS) under real-life conditions using the urinary NNK metabolites 4-(N- methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-(N- methylnitrosamino)-1-(3-pyridyl)but-1-yl]-β-O-D-glucosiduronic acid (NNAL- Gluc). NNK was not detectable (<0.5 ng m-3) in 11 rooms in which smoking did not occur. The mean NNK concentration in 19 rooms in which smoking took place was 17.5 (2.4-50.0) ng m-3. The NNK levels significantly correlated with the nicotine levels (r = 0.856; p < 0.0001). Of the 29 non-smokers investigated, 12 exhibited no detectable NNAL and NNAL-Gluc excretion (< 3 pmol day) in their urine. The mean urinary excretion of NNAL and NNAL-Gluc of the 17 remaining non-smokers was 20.3 (<3-63.2) and 22.9 (<3-90.0) pmol day-1, respectively. Total NNAL excretion (NNAL + NNAL-Gluc) in all non-smokers investigated significantly correlated with the amount of nicotine on personal samplers worn during the week prior to urine collection (r = 0.88; < 0.0001) and with the urinary cotinine levels (r = 0.40; p = 0.038). No correlation was found between NNAL excretion and the reported extent of ETS exposure. Average total NNAL excretion in the non-smokers with detectable NNAL levels was 74 times less than in 20 smokers who were also investigated. The cotinine/total NNAL ratios in urine of smokers (9900) and non-smokers (9300) were similar. This appears to be at variance with the ratios of the corresponding precursors (nicotine/NNK) in mainstream smoke (16 400) and ETS (1000). Possible reasons for this discrepancy are discussed. The possible role of NNK as a lung carcinogen in non-smokers is unclear, especially since NNK exposure in non-smokers is several orders of magnitude lower than the ordinary exposure to exogenous and endogenous N-nitrosamines and the role of NNK as a human lung carcinogen is not fully understood.
Chapter
This chapter describes the aromatic amines, nitroarenes, and heterocyclic aromatic amines. Aromatic amines are important intermediates for the production of polymers, pesticides, rubber chemicals, dyes, pigments, and pharmaceuticals. The toxicity of the aromatic amines has been recognized since the early industrial use of aniline, when the clinical picture of anilism was first seen. Nitroarenes are much more lipophilic than the corresponding arylamines. Monocyclic nitroarenes have a sour to almond odor and taste, which are very prominent for nitrobenzene. The finding that extracts of cooked meats produce a potent response in the Ames test led to the isolation and identification of a number of heterocyclic aromatic amines. These potent mutagens have been shown to be carcinogenic in laboratory animals, and their importance in the etiology of diet-related human cancer is of growing interest.
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Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas, Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens, Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethylamine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/ 4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [H-3]N-hydroxy-ABP) of pancreatic cytosols was high, about two-thirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using P-32-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.
Article
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has a chiral center but the tumorigenicity of the NNAL enantiomers has not been previously examined. In this study, we assessed the relative tumorigenic activities in the A/J mouse of NNK, racemic NNAL, (R)-NNAL, (S)-NNAL and several NNAL metabolites, including [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] b-O-D-glucosiduronic acid [(S)-NNAL-Gluc], 4-(methylnitrosamino)-1(3-pyridyl N-oxide)-1-butanol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran, 4-(3-pyridyl)butane-1,4-diol and 2-(3-pyridyl) tetrahydrofuran. We also quantified urinary metabolites of racemic NNAL and its enantiomers and investigated their metabolism with A/J mouse liver and lung microsomes. Groups of female A/J mice were given a single i.p. injection of 20 mmol of each compound and killed 16 weeks later. Based on lung tumor multiplicity, (R)-NNAL (25.6 K 7.5 lung tumors/mouse) was as tumorigenic as NNK (25.3 K 9.8) and significantly more tumorigenic than racemic NNAL (12.1 K 5.6) or (S)-NNAL (8.2 K 3.3) (P < 0.0001). None of the NNAL metabolites was tumorigenic. The major urinary metabolites of racemic NNAL and the NNAL enantiomers were 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), NNAL-N-oxide and NNAL-Gluc, in addition to unchanged NNAL. Treatment with (R)-NNAL or (S)-NNAL gave predominantly (R)-hydroxy acid or (S)-hydroxy acid, respectively, as urinary metabolites. While treatment of mice with racemic or (S)-NNAL resulted in urinary excretion of (S)-NNAL-Gluc, treatment with (R)-NNAL gave both (R)NNAL-Gluc and (S)-NNAL-Gluc in urine, apparently through the metabolic intermediacy of NNK. (S)-NNAL appeared to be a better substrate for glucuronidation than (R)-NNAL in the A/J mouse. Mouse liver and lung microsomes converted NNAL to products of a-hydroxylation, to NNAL-N-oxide, to adenosine dinucleotide phosphate adducts and to NNK. In lung microsomes, metabolic activation by a-hydroxylation of (R)-NNAL was significantly
Article
A method for the analysis of aromatic amines in tobacco smoke has been developed. The amines from the smoke are trapped in dilute hydrochloric acid and are enriched together with the basic portion, derlvatlzed to pentafluoroproplonamides and determined by gas chromatography with a 63-NI. electron capture detector (detection limit 50 pg aniline/ cigarette). The mainstream smoke of one U.S. 85-mm cigarette without filter tip contained 102 ng of aniline, 61 ng of 2-,3-, and 4-toluidine, 55.8 ng of ethyl- and dimethylaniline, 4.3 ng of 1-naphthylamine, 6.9 ng of 2-,3-, and 4-aminobiphenyl, and 5.8 ng of 2-methyl-l-naphthylamine. Sldestream smoke contained levels of aromatic amines 20 to 68 times' higher than those in the mainstream smoke. The results from smoke analyses of experimental cigarettes support the concept that the nitrate content and the protein content of tobacco are determining factors for the smoke yields of aromatic amines. The possible biologic implications of this study are discussed.
Article
The exposure of non-smokers to the tobacco-specific N-nitrosamine 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a rodent lung carcinogen, was determined in the air of various indoor environments as well as by biomonitoring of non-smokers exposed to environmental tobacco smoke (ETS) under real-life conditions using the urinary NNK metabolites 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-(N-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). NNK was not detectable (<0.5 ng m(-3)) in 11 rooms in which smoking did not occur. The mean NNK concentration in 19 rooms in which smoking took place was 17.5 (2.4-50.0) ng m(-3). The NNK levels significantly correlated with the nicotine levels (r=0.856; p< 0.0001). Of the 29 non-smokers investigated, 12 exhibited no detectable NNAL and NNAL-Gluc excretion (<3 pmol day) in their urine. The mean urinary excretion of NNAL and NNAL-Gluc of the 17 remaining non-smokers was 20.3 (<3-63.2) and 22.9 (<3-90.0) pmol day(-1), respectively. Total NNAL excretion (NNAL+NNAL-Gluc) in all non-smokers investigated significantly correlated with the amount of nicotine on personal samplers worn during the week prior to urine collection (r=0.88; <0.0001) and with the urinary cotinine levels (r=0.40; p=0.038). No correlation was found between NNAL excretion and the reported extent of ETS exposure. Average total NNAL excretion in the non-smokers with detectable NNAL levels was 74 times less than in 20 smokers who were also investigated. The cotinine/total NNAL ratios in urine of smokers (9900) and non-smokers (9300) were similar. This appears to be at variance with the ratios of the corresponding precursors (nicotine/NNK) in mainstream smoke (16400) and ETS (1000). Possible reasons for this discrepancy are discussed. The possible role of NNK as a lung carcinogen in non-smokers is unclear, especially since NNK exposure in non-smokers is several orders of magnitude lower than the ordinary exposure to exogenous and endogenous N-nitrosamines and the role of NNK as a human lung carcinogen is not fully understood.