TOXICOLOGICAL SCIENCES 86(1), 141–151 (2005)
doi:10.1093/toxsci/kfi112
Advance Access publication February 16, 2005
Phenols, Quinolines, Indoles, Benzene, and 2-Cyclopenten-1-ones are
Oviductal Toxicants in Cigarette Smoke
Karen Riveles,* R. Roza,† and P. Talbot†,1
*Graduate Program in Environmental Toxicology and †Department of Cell Biology and Neuroscience, University of California Riverside, Riverside,
California 92521
Received December 10, 2004; accepted February 8, 2005
Previously, we showed that pyridines and pyrazines in cigarette
smoke inhibit oviductal functioning in vitro in nanomolar and
picomolar doses. The purpose of this study was to determine the
lowest observable adverse effect levels (LOAELs) of phenols,
quinolines, indoles, benzene, and 2-cyclopenten-1-ones found in
mainstream smoke solutions on ciliary beat frequency, oocyte
pickup rate, and infundibular smooth muscle contraction using
the hamster oviduct. After solid phase extraction, mainstream
smoke solution fractions were tested in the oviductal assays. The
active fractions were analyzed using gas chromatography-mass
spectrometry to identify individual chemicals. Using this approach, benzene, eleven phenolic, two indole, two quinoline, and
two 2-cyclopenten-1-one derivatives were identified in the active
fractions. Commercially available authentic standards of the
identified compounds were tested in dose-response studies on
hamster oviducts. The LOAELs were determined for each
compound using the ciliary beat frequency, oocyte pickup rate,
and infundibular smooth muscle contraction rate assays. Indole,
the compound with the highest potency, showed inhibition of
ciliary beat frequency (1013 M), oocyte pickup rate (1014 M),
and infundibular smooth muscle contraction rate (1015 M) in
femtomolar doses. All of the other compounds tested, except
phenol, which only showed inhibition at millimolar concentrations, were inhibitory in picomolar, nanomolar, or micromolar
concentrations. Derivitization of phenol increased its toxicity in
the oviductal assays, especially when a methyl or ethyl group was
substituted on the fourth position. The indoles, quinolines, and
four phenolic compounds had both high potencies and efficacies in
the oviductal assays.
Key Words: cigarette smoke; oviduct; oocyte pickup; phenols.
Numerous epidemiological studies have linked cigarette
smoking with reproductive problems in women. For example,
an association has been observed between women who smoke
and increased incidences of ectopic pregnancy, tubal infertility, and spontaneous abortion (Castles et al., 1999; Mueller
and Ciervo, 1998; Saraiya et al., 1998). These problems could
be due to an effect on the oviduct, and indeed, recent in vivo
1
To whom correspondence should be addressed. Fax: (951) 827-4286.
E-mail: [email protected].
and in vitro studies have shown an effect on oviductal
functioning by cigarette smoke (DiCarlantonio and Talbot,
1999; Knoll et al., 1995; Knoll and Talbot, 1998; Magers
et al., 1995; Riveles et al., 2003). Factors such as cigarette
smoke that impair oviductal functioning can diminish or
prevent fertility (Florek and Marszalek, 1999; Shiverick and
Salafia, 1999).
Inhalation of mainstream (MS) or sidestream (SS) smoke by
hamsters, at serum cotinine levels that were within the ranges
found in active and passive human smokers, adversely affected
the ultrastructure of the oviductal epithelium (Magers et al.,
1995). Decreases in the ratio of ciliated to secretory cells in the
ampulla were also observed in these hamsters (Magers et al.,
1995). In another study on hamsters, inhalation of MS and SS
smoke slowed embryo transport, and 75% of the embryos were
retained in the oviduct (DiCarlantonio and Talbot, 1999).
Moreover, oviductal contractions were inhibited differentially
in the ampulla and isthmus in vivo after smoke exposure,
suggesting that embryo transport rates were slowed by smooth
muscle contraction (DiCarlantonio and Talbot, 1999). Studies
have also shown that cigarette smoke exposure slowed the rate
of smooth muscle contraction in humans (Neri and Eckerling,
1969) and in rabbits (Ruckebusch, 1975) after in vivo exposure.
In vitro studies have also shown that MS and SS smoke
solutions adversely affect proper functioning of the oviduct.
MS and SS smoke solutions made with University of Kentucky
2R1 research cigarettes inhibited oocyte pickup rate in a dosedependent manner in explants of hamster oviducts in vitro
(Knoll et al., 1995; Knoll and Talbot, 1998). Ciliary beat
frequency decreased in MS smoke solutions; however, SS
produced no change or stimulated ciliary beating (Knoll et al.,
1995; Knoll and Talbot, 1998). The gas and particulate phases
of the smoke solutions were also tested in the oocyte pickup
rate assay. Oocyte pickup rate was more sensitive to the gas
than the particulate phase of MS and SS smoke solutions
(Knoll and Talbot, 1998).
Several studies have been done to identify the toxicants in
cigarette smoke that affect the oviduct. For example, oral
administration of nicotine inhibited oviductal contractions in
Rhesus monkeys (Neri and Marcus, 1972). Intravenous
Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For Permissions, please email: [email protected]
142
RIVELES, ROZA, AND TALBOT
administration of nicotine delayed rat embryo cleavage, reduced embryo cell number, and reduced oviductal blood flow
(Mitchell and Hammer, 1985). In another study, other individual smoke components (potassium cyanide, acrolein,
phenol, acetaldehyde, and formaldehyde), inhibited ciliary
beat frequency in the hamster oviduct in vitro; however, only
cyanide was present in cigarette smoke solutions in sufficiently
high concentrations to account for the effects seen in vitro
(Talbot et al., 1998).
As a result of earlier epidemiological and model systems
studies, our lab developed an assay using the hamster infundibulum in vitro to study oviductal functioning and we have
been determining the compounds in smoke that affect oocyte
pickup rate, ciliary beat frequency, and infundibular smooth
muscle contraction rate. Pyridine compounds, which are added
directly to tobacco and are generated indirectly by combustion
processes associated with burning of the cigarette, have been
identified in MS and SS smoke solutions and adversely affect
hamster oviductal functioning in vitro (Riveles et al., 2003).
Twelve pyridine compounds, including nicotine, were tested on
oviductal functioning in vitro, measuring ciliary beat frequency, oocyte pickup rate, and oviductal smooth muscle
contraction rate (Riveles et al., 2003). Pyridine derivatives
with single ethyl or methyl substitutions (2-ethylpyridine,
3-ethylpyridine, 2-methylpyrdine, and 4-methylpyridine) were
inhibitory at picomolar doses for all three parameters measured
(Riveles et al., 2003). Nornicotine and b-nicotyrine, which are
similar in structure to nicotine, were inhibitory in nanomolar
doses in the bioassays, while nicotine was only inhibitory at
102 M.
In the second study of the series, pyrazine compounds,
which are additives in food, tobacco, and cosmetics, and are
combustion products of tobacco, were identified in both MS
and SS smoke solutions and tested on the hamster oviduct
in vitro (Riveles et al., 2004). Six of the seven pyrazine
compounds inhibited oviductal functioning at picomolar or
nanomolar doses in vitro. Several pyrazine compounds with
single ethyl or methyl substitutions (2-ethylpyrazine and
2-methylpyrazine), as well as unsubstituted pyrazine, inhibited ciliary beat frequency, oocyte pickup rate, and
smooth muscle contraction rate at picomolar doses (Riveles
et al., 2004).
The purpose of this study was to test the hypothesis that there
are additional classes of components identified in MS and SS
smoke solutions that adversely affect ciliary beat frequency,
oocyte pickup rate, and infundibular smooth muscle contraction rate. To test this hypothesis, benzene, indole, 5-methylindole,
quinoline, isoquinoline, 2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and eleven phenolic compounds were individually tested in dose-response experiments on the hamster
oviduct in vitro, measuring ciliary beat frequency, oocyte
pickup rate, and smooth muscle contraction rate. Our data
show that several compounds were inhibitory in as low as
femtomolar or picomolar doses.
MATERIALS AND METHODS
Animals, media, and authentic standards. Female golden hamsters
(Mesocricetus auratus) purchased from Harlan (San Diego, CA) were
maintained on a 14L:10D cycle in a room at 26°C. Food was administered
ad libitum. Hamsters were cycled daily by checking for a vaginal discharge,
which occurs on day 1 of their estrous cycle. Female golden hamsters were
induced to superovulate by intraperitoneal injection with 25 International Units
(IU) of pregnant mare’s serum gonadotropin (PMSG) (CalBiochem, La Jolla,
CA.) at 10 A.M. on day 1 of their estrous cycle, followed by 25 IU of human
chorionic gonadotropin (hCG) (Sigma Chemical Co., St. Louis, MO) on day
3 of the estrous cycle. Hamsters were sacrificed with CO2 on day 4,
approximately 12 h after the hCG injection, and oviducts and ovaries were
removed and separated. The two ovaries were isolated in Earle’s Balanced Salt
Solution supplemented with sodium bicarbonate, HEPES, and 0.1% BSA at
pH ¼ 7.4 (EBSS-H). The expanded follicles were poked with a dissecting
needle to release the oocyte cumulus complexes (OCCs). Approximately 7–15
mature expanded OCCs were recovered from each ovary. The two infundibula
were dissected from the oviducts and placed into perfusion chambers
containing EBSS-H. The ampullas were left attached to the infundibula and
placed into holding pipettes so the infundibula remained stationary throughout
the experiment. EBSS-H was used as the control solution for all experiments.
The protocol for the use of hamsters in this study was approved by the campus
animal use committee.
2-Cyclopenten-1-one (98%), 2,4-dimethylphenol (98%), 2-ethylphenol
(99%), 4-ethylphenol (99%), indole (98%), 2-methoxyphenol (98%),
p-methoxyphenol (99%), 3-methyl-2-cyclopenten-1-one(97%), 5-methylindole
(99%), 2-methylphenol (99þ%), 4-methylphenol (99%), and quinoline (98%)
were purchased from Aldrich Chemical Company (Milwaukee, WI). 2,6Dimethoxyphenol (96%) and isoquinoline were purchased from City Chemical
LLC (West Haven, CT.). Hydroquinone (99%) was purchased from VWR
(Brisbane, CA). Benzene (99%) and phenol (99%) were purchased from Fisher
Scientific (Pittsburgh, PA).
Preparation of smoke solutions. Mainstream (MS) smoke solutions were
made in EBSS-H using 2R1 research-grade cigarettes (University of Kentucky,
Louisville, KY) that were smoked on a puffer box built at the University of
Kentucky (Knoll et al., 1995; Knoll and Talbot, 1998). This method has been
described and published previously in detail (Knoll et al., 1995; Knoll and
Talbot, 1998; Riveles et al., 2003). MS smoke solutions were made from 60
puffs of MS smoke pushed through 10 ml of EBSS-H, which equals 6 puff
equivalents.
Solid-phase extraction and gas-chromatography-mass spectrometry
(GC-MS) of smoke solutions. Bond Elut solid phase extraction (SPE)
cartridges, 3 cc, 500 g capacity (Phenomenex, Torrance, CA) were used to
fractionate smoke solutions and concentrate chemicals that inhibit oviductal
functioning. A variety of nonpolar, polar, and anion and cation exchange
cartridges were used: NH2, 2OH, CN, CBA, SCX, SAX, C18, C8, C2, CH, SI,
and PH. The protocol used to screen the cartridges for their ability to bind
oviductal toxicants in smoke solutions has been described in detail previously
(Riveles et al., 2003). MS smoke solutions were passed through the cartridges,
and the individual fractions and corresponding cartridge eluates were collected
and tested in the oviductal bioassays measuring ciliary beat frequency, oocyte
pickup rate, and smooth muscle contraction rate (Riveles et al., 2003).
Four cartridges (C8, CN, PH, and NH2) were particularly effective at
retaining the oviductal toxicants, and their eluates were characterized using
GC-MS. A Hewlett Packard 5890 GC interfaced to an HP-5971A MSD
quadrupole mass selective detector with a Zebron ZB1701 cyanopropyl phenyl
column 30 m 3 0.32 mm with 1-lm phase thickness from Phenomenex
(Torrance, CA.) was used as described previously in detail (Riveles et al.,
2003). The eluate sample (2 ll) was injected directly into the GC, and
compounds were identified using the mass spectrometry data matched to mass
spectral library entries. Compound identities were confirmed using authentic
standards and by matching both mass spectra and retention times.
143
SMOKE TOXICANTS INHIBIT OVIDUCT FUNCTION
Oviductal assays: ciliary beat frequency, oocyte pickup rate, and
infundibular smooth muscle contraction rate. Ciliary beat frequency, oocyte
pickup rate, and smooth muscle contraction rate were measured using hamster
oviduct explants in vitro in a bioassay developed in our lab and described
previously in detail (Riveles et al., 2003). A single experiment was defined as
the treatment of one infundibulum with the control medium (EBSS-H with
0.1% BSA) and a single dose of the test chemical diluted in EBSS-H for a
5-min exposure period. The mean ± standard deviation of six to ten measurements was made on an infundibulum for each treatment group. For each
parameter, a region of the infundibulum was selected, and measurements for
both the control and treated groups were performed in the same region.
Each parameter was measured using the same experimental setup with
a Wild 5A stereoscopic microscope and a video image capture system as
described previously (Riveles et al., 2003). Ciliary beat frequency was
measured using the video capture system and expressed as number of beats
per second. Oocyte pickup rate was performed by manually placing an oocyte
cumulus complex (OCC) onto the infundibulum and measuring the speed at
which the OCC traversed a defined path expressed as microns per second.
Infundibular smooth muscle contractions were also measured using the video
image capture system. The distance of the contractions was measured directly
on the computer monitor using a ruler as described previously (Riveles et al.,
2003). Frequency was measured as the number of times the infundibulum
contracted per minute. Smooth muscle contraction rate was calculated and
expressed as microns per minute.
Determination of LOAELs and efficacies. To determine LOAELs for
each compound tested in each bioassay, a preliminary screen was done using
one infundibulum per dose group (1016–101 M). For each infundibulum, 6–
10 control, treatment, and recovery measurements were taken, and the means
were compared using a one-way ANOVA. The lowest significant inhibitory
dose provided an estimate of the LOAELs for each test compound in each
assay. Since the estimated LOAELs were based on only one infundibulum per
dose, additional experiments were performed at the estimated NOAEL and
LOAEL doses to increase the sample size to n ¼ 4 infundibula for all of the
tested compounds in each bioassay.
Using the initial dose-response curves, the efficacy values were estimated
for each test compound in each assay. Efficacies were determined by finding the
point on the curve that reached a maximum percentage of inhibition and then
leveled off over two or more consecutive doses. These efficacy values are
estimations based on a limited dose selection and a sample size of only one
infundibulum. A maximum percentage of inhibition was not reached in all
cases based on the doses selected, in which case efficacies were indicated as
greater than or equal to the highest percentage of inhibition observed for that
compound in that assay at the highest dose tested.
Statistical analysis. The statistical significance of the results was evaluated in the oocyte pickup rate, ciliary beat frequency, and infundibular muscle
contraction rate assays using a one sample t-test to compare the control (n ¼ 4
infundibula) set to 100% to the mean at the estimated LOAEL dose (n ¼ 4
infundibula) for each test compound in each bioassay (Fig. 7) with Instat
(Graphpad, San Diego, CA). Means were considered to be significantly
different for p < 0.05.
RESULTS
Phenols, Indoles, Quinolines, Benzene, and 2-Cyclopenten1-Ones Identified in MS Smoke Solutions
A preliminary screen of 12 solid phase extraction cartridges
revealed that four of the cartridges (C8, CN, PH, and NH2)
retained chemicals in MS smoke solutions that were inhibitory
in the ciliary beat frequency, oocyte pickup rate, and smooth
muscle contraction rate assays. Phenols, indoles, quinolines,
TABLE 1
Individual Chemicals Identified in the Inhibitory Eluates from
Four Solid Phase Extraction Cartridges Loaded with Mainstream
Smoke Solution
Chemicals
CAS #
Phenol
108–95–2
2-Methylphenol
95–48–7
4-Methylphenol
106–44–5
2,4-Dimethylphenol
105–67–9
2,5 Dimethylphenol
95–87–4
2-Methoxyphenol
90–05–1
4-Methoxyphenol
150–76–5
2,6-Dimethoxyphenol
91–10–1
2-Ethylphenol
90–00–6
4-Ethylphenol
123–07–9
Hydroquinone
123–31–9
Indole
120–72–9
5-Methylindole
614–96–0
Quinoline
91–22–5
Isoquinoline
119–65–3
Benzene
71–43–2
2-Cyclopenten-1-one
930–30–3
3-Methyl-2-cyclopenten-1-one 2758–18–1
MW
(g)
94
108
108
122
122
124
124
154
122
122
110
117
131
129
129
78
82
96
Cartridge eluatesa
C8
PH
CN
NH2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
a
Solid phase extraction cartridges are C8 (Octyl), which is an 8-carbon
straight-chain hydrocarbon; CN (cyanopropyl); PH (phenyl); and NH2
(aminopropyl).
benzene, and 2-cyclopenten-1-ones were subsequently identified in the eluates of these four cartridges using GC-MS (Table
1). Commercially available authentic standards of the identified
compounds from Table 1 were purchased and tested in doseresponse studies on the oviduct to determine their lowest
observable adverse effect levels (LOAELs) in the oviductal
bioassays.
Dose-Response, LOAELs, and Estimated Efficacies of the
Phenolic Compounds in the Oviductal Bioassays
Phenol and 10 phenolic derivatives (2-methylphenol, 4methylphenol, 2-ethylphenol, 4-ethylphenol, 2,4-dimethylphenol,
2,5-dimethylphenol, 2-methoxyphenol, 4-methoxyphenol,
2,6-dimethoxyphenol, and hydroquinone) were tested in
dose-response studies on the hamster oviduct based on a 5min exposure in the ciliary beat frequency, oocyte pickup rate,
and smooth muscle contraction rate assays. For each phenolic
compound tested, an initial screen of doses, ranging from 1013
M to 101 M was performed for a single infundibulum per dose
group. For the parent compound, phenol, the dose range was
extended to 10 M. Dose-response curves for the ciliary beat
frequency, oocyte pickup rate, and smooth muscle contraction
rate assays were created based on these data (Figs. 1–3).
LOAELs were estimated from the dose-response curves and
then confirmed using an increased sample size (n ¼ 4
144
RIVELES, ROZA, AND TALBOT
40
35
Percent Inhibition
30
25
20
Phenol
4-Methylphenol
4-Ethylphenol
2,5-Dimethylphenol
4-Methoxyphenol
Hydroquinone
120
2-Meylphenol
2-Ethylphenol
2,4-Dimethylphenol
2-Methoxyphenol
2,6-Dimethoxyphenol
100
Percent Inhibition
Phenol
2-Methylphenol
4-Methylphenol
2-Ethylphenol
4-Ethylphenol
2,4-Dimethylphenol
2,5-Dimethylphenol
2-Methoxyphenol
4-Methoxypheno
2,6-Dimethoxyphenol
Hydroquinone
80
60
40
15
20
10
0
10-1310-1210-1110-1010-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 101 102 103
5
Concentration (M)
0
10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 101 102 103
Concentration (M)
FIG. 1. Dose-response curves for phenolic compounds in the ciliary beat
frequency assay. Dose-response curves for ciliary beat frequency are based on
an initial screen of phenolic compounds with a single infundibulum per dose
group. Each value is the mean ± standard deviation of 6–10 measurements.
infundibula) for each of the bioassays. The confirmed LOAELs
for the phenolic compounds (n ¼ 4) are summarized in Table 2.
Figure 7 has the mean percent inhibition and standard deviation
at each LOAEL for each of the bioassays. The estimated
efficacies were determined from the initial dose-response
curves as explained in the Materials and Methods.
80
Phenol
4-Methylphenol
4-Ethylphenol
2,5-Dimethylphenol
4-Methoxyphenol
Hydroquinone
FIG. 3. Dose-response curves for phenolic compounds in the smooth
muscle contraction rate assay. Dose-response curves for smooth muscle
contraction rate are based on an initial screen of phenolic compounds with
a single infundibulum per dose group. Each value is the mean ± standard
deviation of 6–10 measurements.
For the ciliary beat frequency assay, the dose-response
curves were similar for most of the phenolic compounds,
except for 2-ethylphenol and phenol, which were inhibitory at
higher doses (Fig. 1). It is interesting that all of the phenolic
derivatives were more potent than phenol itself. Additional
infundibula were tested at the estimated LOAEL doses for
confirmation. The LOAELs for the phenolic compounds
ranged from 1012 M to 101 M (Table 2). 4-Methylphenol
(1012 M), 4-ethylphenol (1011 M), 2,6-dimethoxyphenol
(1010 M), and hydroquinone (1010 M) inhibited ciliary beat
frequency at picomolar doses. 2-Methylphenol (109 M),
2,4-dimethylphenol (109 M), 2-methoxyphenol (108 M),
2-Methylphenol
2-Ethylphenol
2,4-Dimethylphenol
2-Methoxyphenol
2,6-Dimethoxyphenol
TABLE 2
LOAELs for Phenol Derivatives in the Oviductal Assays
Percent Inhibition
70
LOAELs (M)a
60
50
Chemical
40
30
20
10
0
10-1310-12 10-1110-10 10-9 10-810-7 10-6 10-5 10-4 10-3 10-2 10-1 101 102 103
Concentration (M)
FIG. 2. Dose-response curves for phenolic compounds in the oocyte
pickup rate assay. Dose-response curves for oocyte pickup rate are based on an
initial screen of phenolic compounds with a single infundibulum per dose
group. Each value is the mean ± standard deviation of 6–10 measurements.
Phenol
2-Methylphenol
4-Methylphenol
2-Ethylphenol
4-Ethylphenol
2,4-Dimethylphenol
2,5-Dimethylphenol
2-Methoxyphenol
4-Methoxyphenol
2,6-Dimethoxyphenol
Hydroquinone
a
Ciliary beat
frequency
Oocyte
pickup rate
Contraction
rate
101
109
1012
105
1011
109
108
108
107
1010
1010
102
1011
1011
108
1012
1010
107
1010
108
1011
1010
102
1011
1011
107
1012
109
106
108
107
109
1010
LOAEL is lowest observable adverse effect level.
SMOKE TOXICANTS INHIBIT OVIDUCT FUNCTION
2,5-dimethylphenol (108 M), and 4-methoxyphenol (107 M)
inhibited ciliary beat frequency at nanomolar doses. 2Ethylphenol (105 M) inhibited ciliary beat frequency at
micromolar doses. Phenol had the highest LOAEL (101 M).
The substitution of an ethyl or methyl group in the fourth
position on the phenol moiety compared to the substitution on
the second position increased its potency by a million-fold and
one-thousand-fold, respectively.
For the oocyte pickup rate assay, the dose-response curves were
similar for six of the phenolic compounds (2-methylphenol,
2,4-dimethylphenol, 4-ethylphenol, 2-methoxyphenol, 2,6dimethoxyphenol, and hydroquinone) (Fig. 2). The doseresponse curves for 2-ethylphenol, 2,5-dimethylphenol, and 4methoxyphenol showed inhibition at higher doses. All of the
phenolic derivatives were more potent than phenol itself, which
had a LOAEL for oocyte pickup rate (102 M), similar to the
ciliary beat frequency assay (101 M). Additional infundibula
were tested at the estimated LOAEL doses for confirmation. The
LOAELs for the phenolic compounds in the oocyte pickup rate
assay ranged from 1012 M to 102 M (Table 2). 4-Ethylphenol,
(1012 M), 2-methylphenol (1011 M), 4-methylphenol (1011
M), 2,6-dimethoxyphenol (1011 M), 2,4-dimethylphenol (1010
M), 2-methoxyphenol (1010 M), and hydroquinone (1010 M)
inhibited oocyte pickup rate in picomolar doses. 2-Ethylphenol
(108 M), 4-methoxyphenol (108 M), and 2,5-dimethylphenol
(107 M) inhibited oocyte pickup rate at nanomolar doses.
Phenol had the highest LOAEL (102 M) (Table 2).
For the smooth muscle contraction rate assay, all of the
derivatives were more potent than phenol (102 M) (Fig. 3).
The dose-response curves were similar for most of the phenolic
compounds, except for 2-methylphenol, 4-methylphenol, and
phenol (Fig. 3). The dose-response curves for 2-ethylphenol
(107 M), 4-methoxyphenol (107 M), and 2,5-dimethylphenol
(106 M) had LOAELs at higher doses in this assay, as was the
case in the oocyte pickup rate assay (108, 108 M, and 107,
respectively). Additional infundibula were tested at the estimated LOAEL doses for confirmation. For the smooth muscle
contraction rate assay, the LOAELs for the phenolic compounds ranged from 1012 M to 102 M (Fig. 3). 4-Ethylphenol
(1012 M), 2-methylphenol (1011 M), 4-methylphenol (1011
M), and hydroquinone (1010 M) inhibited smooth muscle
contraction rate at picomolar doses. 2,4-Dimethylphenol (109
M), 2,6-dimethoxyphenol (109 M), 2-methoxyphenol (108
M), 2-ethylphenol (107 M), and 4-methoxyphenol (107 M)
inhibited smooth muscle contraction rate at nanomolar doses.
2,5-Dimethylphenol (106 M) inhibited smooth muscle contraction rate at micromolar doses. The LOAEL for phenol was
the highest at millimolar doses (102 M) (Fig. 3).
Using the initial dose-response curves, the efficacies for
phenol and several of its derivatives were estimated as
described in the Materials and Methods. The estimated
efficacies of phenol in the ciliary beat frequency, oocyte pickup
rate, and smooth muscle contraction rate assays were 22%,
68%, and 100%, respectively (Figs. 1–3). The chemicals
145
having estimated efficacies in the ciliary beat frequency assay
that were lower than phenol were 2,4-dimethylphenol (7%)
and 4-methylphenol (16%) (Fig. 1). 2-Methylphenol (25%),
2,5-dimethylphenol (28%), 2-ethylphenol (32%), and hydroquinone (37%) had estimated efficacies in the ciliary beat
frequency assay that were higher than phenol (Fig. 1). The
efficacies for 2-methoxyphenol (9%), 4-methoxyphenol
(11%), 2,6-dimethoxyphenol (14%), and 4-ethylphenol
(19%) were estimated based on the initial dose-response
curves (Fig. 1).
For the oocyte pickup rate assay, from the dose-response
curve, the efficacy for phenol was 68% (Fig. 2). The estimated
efficacy of 4-methylphenol (6%) was lower than for phenol in the
oocyte pickup rate assay (Fig. 2). The efficacies for 4-ethylphenol
(41%), 2-methoxyphenol (41%), 2-methylphenol (42%),
4-methoxyphenol (50%), 2,6-dimethoxyphenol (52%),
hydroquinone (63%), 2,4-dimethylphenol (64%), 2,5dimethylphenol (67%), and 2-ethylphenol (75%) were
estimated for the oocyte pickup rate assay based on the doseresponse curves (Fig. 2). The efficacies in the oocyte pickup
rate assay were higher than for the ciliary beat frequency assay
in most cases.
In the smooth muscle contraction rate assay, the estimated
efficacy of phenol was 100% (Fig. 3). The estimated efficacies
of 4-methylphenol (6%) and 2,6-dimethoxyphenol (66%) were
lower than phenol in the smooth muscle contraction rate
assay (Fig. 3). 4-Ethylphenol (100%) and 4-methoxyphenol
(100%) had efficacies similar to phenol (Fig. 3). The efficacies
for 2-methylphenol (16%), 2-methoxyphenol (30%), 2ethylphenol (39%), 2,4-dimethylphenol (65%), and hydroquinone (71%) were estimated in the smooth muscle
contraction rate assay based on the initial dose-response curves
(Fig. 3).
Dose-Response, LOAELs, and Estimated Efficacies of
Indoles, Quinolines, Benzene, and 2-Cyclopenten-1-ones
in the Oviductal Bioassays
An initial screen of doses ranging from 1016 M to 104 M
was used to test indole, 5-methylindole, quinoline, isoquinoline, benzene, 2-cyclopenten-1-one, and 3-methyl-2-cylcopenten-1-one in the three bioassays with a single
infundibulum per dose group. Dose-response curves for the
ciliary beat frequency, oocyte pickup rate, and smooth muscle
contraction rate assays were created based on these data (Figs.
4–6). LOAELs were estimated from the dose-response curves
and then confirmed using an increased sample size (n ¼ 4
infundibula). The confirmed LOAELs for these compounds
(n ¼ 4) are summarized in Table 3. Figure 7 has the mean
percent inhibition and standard deviation at each LOAEL for
each of the bioassays.
For the ciliary beat frequency assay, the dose-response curves
were similar for the indoles and quinolines (Fig. 4). Benzene, 2cyclopenten-1-one, and 3-methyl-2-cyclopenten-1-one showed
146
RIVELES, ROZA, AND TALBOT
40
5-Methylindole
Quinoline
90
Isoquinoline
Benzene
80
Percent Inhibition
50
Percent Inhibition
Indole
Indole
5-Methylindole
Quinoline
Isoquinoline
Benzene
2-Cyclopenten-1-one
3-Methyl-2-cyclopenten-1-one
60
30
20
10
2-Cyclopenten-1-one
70
3-Methyl-2-cyclopenten-1-one
60
50
40
30
20
10
0
10
-16
10
-15
10
-14
10
-13
-12
-11
-10
10
10 10
10
Concentration (M)
-9
10
-8
10 -7 10 -6
0
10-16 10-15 10-14 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5
Concentration (M)
FIG. 4. Dose-response curves for the indoles, quinolines, benzene, and 2cyclopenten-1-ones in the ciliary beat frequency assay. Dose-response curves
for the ciliary beat frequency assay are based on an initial screen of the compounds with a single infundibulum per dose group. Each value is the mean ±
standard deviation of 6–10 measurements.
FIG. 6. Dose-response curves for the indoles, quinolines, benzene, and 2cyclopenten-1-ones in the smooth muscle contraction rate assay. These are
based on an initial screen of the compounds with a single infundibulum per
dose group. Each value is the mean ± standard deviation of 6–10 measurements.
inhibition at higher doses and were the least potent in this
group of chemicals (Fig. 4). Additional infundibula were tested
to confirm the LOAELs for each compound in the ciliary beat
frequency assay. The LOAELs ranged from 1013 M to 108 M
(Table 3). Indole (1013 M), quinoline (1013 M), isoquinoline
(1012 M), and 5-methylindole (1010 M) inhibited ciliary beat
frequency at femtomolar or picomolar doses. Benzene (108
M) and 2-cyclopenten-1-one (108 M) inhibited ciliary beat
frequency at nanomolar doses. The LOAEL for 3-methyl-2cyclopenten-1-one in the ciliary beat frequency was not
determined with the dose range used (Fig. 4).
For the oocyte pickup rate assay, the dose-response curve for
indole showed the highest potency of all the chemicals tested
(Fig. 5). The dose-response curves for 5-methylindole, quin-
oline, and isoquinoline in the oocyte pickup rate assay were
similar to the ciliary beat frequency assay. Benzene and 3methyl-2-cyclopenten-1-one were the least potent in this assay
as was the case in the ciliary beat frequency assay (Fig. 5).
Additional infundibula were tested to confirm the LOAELs for
each compound in the oocyte pickup rate assay (Fig. 7). The
LOAELs for this group of compounds ranged from 1014 M to
107 M (Table 3). Indole (1014 M), isoquinoline (1013 M),
quinoline (1011 M), 5-methylindole (1011 M), and 3-methyl2-cyclopenten-1-one (1010 M) inhibited oocyte pickup rate
at femtomolar or picomolar doses. Oocyte pickup rate was
inhibited by 2-cyclopenten-1-one (109 M) and benzene (107
M) at nanomolar doses.
Indole
5-Methylindole
Quinoline
Isoquinoline
Benzene
2-Cyclopenten-1-one
3-Methyl-2-cyclopenten-1-one
80
Percent Inhibition
70
TABLE 3
LOAELs for Indoles, Quinolines, Benzene and 2-Cyclopenten-1ones in the Oviductal Assays
60
LOAELs (M)a,b
50
40
Chemical
Ciliary beat
frequency
Oocyte
pickup rate
Contraction
rate
1013
1010
1013
1012
1011
108
X
1014
1011
1011
1013
1012
107
1010
1015
1010
1011
1013
1012
106
1010
30
20
10
0
10-16 10-15 10-14 10-13 10-1210-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
FIG. 5. Dose-response curves for the indoles, quinolines, benzene, and 2cyclopenten-1-ones in the oocyte pickup rate assay. These are based on an
initial screen of the compounds with a single infundibulum per dose group.
Each value is the mean ± standard deviation of 6–10 measurements.
Indole
5-Methylindole
Quinoline
Isoquinoline
Benzene
2-Cyclopenten-1-one
3-Methyl-2-cyclopenten-1-one
a
LOAEL is lowest observable adverse effect level.
‘‘X’’ means that the LOAEL could not be determined at the highest dose
tested for this assay.
b
147
SMOKE TOXICANTS INHIBIT OVIDUCT FUNCTION
Ciliary Beat Frequency
Percent Inhibition
35
**
30
25
20
**
**
**
15
**
**
**
**
**
**
10
**
*
5
**
*
*
*
*
0
2m
e p
4- thy hen
m l
et ph ol
h
e
2- yl p no
l
et
hy hen
2, 4-e l p ol
4h
t
h
e
d
2, im yl p nol
5- et
di hy hen
m l
o
2- eth phe l
m
et yl p nol
4 h
h
2, -m ox en
6- et y
o
di ho ph l
m x e
et y no
ho ph l
x e
hy y p no
dr he l
oq n
ui ol
no
5n
m
et in e
hy do
l i le
n
qu do
l
i
3so ino e
m
qu lin
et 2in e
hy cy
ol
l-2 clo
-c p be ine
yc en nz
lo te en
pe n- e
nt 1-o
en n
-1 e
-o
ne
A.
Oocyte Pickup Rate
Percent Inhibition
80
70
60
50
40
30
20
10
0
**
**
**
*
*
**
**
**
**
**
Estimated Efficacies for Indoles, Quinolines,
2-Cyclopenten-1-ones, and Benzene
**
**
*
**
**
**
**
2m
e p
4- thy hen
m l
et ph ol
h
e
2- yl p no
l
et
hy hen
2, 4-e l p ol
4h
t
e
d h
2, im yl p nol
5- et
di hy hen
m l
o
2- eth ph l
e
m
et yl p no
h
4
h l
2, -m ox en
6- et y
o
di ho ph l
m x e
et y no
p
ho h l
x e
hy y p no
dr he l
oq n
ui ol
no
5n
m
et in e
hy do
l i le
n
qu do
is ino le
3o
m
qu lin
et 2in e
hy cy
ol
l-2 clo
-c p be ine
yc en nz
lo te en
pe n- e
nt 1-o
en n
-1 e
-o
ne
B.
**
Infundibular Smooth Muscle Contraction Rate
Percent Inhibition
70
**
60
**
40
**
**
30
20
10
**
**
**
*
*
*
**
*
*
**
*
0
2m
e p
4- thy hen
m l
et ph ol
h
e
2- yl p no
l
et
hy hen
2, 4-e l p ol
4h
t
e
d h
2, im yl p nol
5- et
di hy hen
m l
o
2- eth ph l
e
m
et yl p no
4 h
h l
2, -m oxy en
6- et
o
di ho ph l
m x en
et y
o
p
ho h l
x e
hy y p no
dr he l
oq n
ui ol
no
5m
n
et in e
hy do
l i le
n
qu do
is ino le
3oq l
m
et 2ui ine
hy cy
no
l-2 clo
lin
b
-c p
e
e
yc n nz e
lo te en
pe n- e
nt 1-o
en n
-1 e
-o
ne
C.
**
**
**
50
For the smooth muscle contraction rate assay, indole showed
the highest potency of all the chemicals tested, as was the case
for oocyte pickup rate (Fig. 6). The dose-response curves for 5methylindole, quinoline, isoquinoline 2-cyclopenten-1-one,
and 3-methyl-2-cyclopenten-1-one were similar for the smooth
muscle contraction rate assay. The dose-response curve for
benzene showed little potency in this assay, as was the case for
the ciliary beat frequency and oocyte pickup rate assays (Fig.
6). Additional infundibula were tested to confirm the LOAELs
for each compound in the smooth muscle contraction rate
assay (Table 3). Indole (1015 M) and isoquinoline (1013 M)
inhibited smooth muscle contraction rate at femtomolar
doses. Quinoline (1011 M), 5-methylindole (1010 M), and
3-methyl-2-cyclopenten-1-one (1010 M) inhibited smooth
muscle contraction rate at picomolar doses. Smooth muscle
contraction rate was inhibited by 2-cyclopenten-1-one (109
M) at nanomolar doses and by benzene (106 M) at micromolar
doses (Fig. 6).
FIG. 7. Percent inhibition at the LOAEL dose for ciliary beat frequency,
oocyte pickup rate and smooth muscle contraction rate. Ciliary beat frequency
(A), oocyte pickup rate (B), and smooth muscle contraction rate (C) are
significantly inhibited by phenols, indoles, quinolines, benzene, and 2-cyclopenten-1-ones. The percent inhibition at the LOAEL dose is expressed as the
mean ± standard deviation of four infundibula. Statistical significance was
determined using a one-sample t-test, where * p < 0.05, ** p < 0.01, and
*** p < 0.001.
Using the initial dose-response curves, the efficacies for the
indoles, quinolines, 2-cyclopenten-1-ones, and benzene were
estimated as described in Materials and Methods. For the
ciliary beat frequency assay, the efficacies for indole (25%)
and 5-methylindole (15%) could not be compared (Fig. 4).
The efficacy of isoquinoline in the ciliary beat frequency
assay was 27%; however, the efficacy of quinoline could only
be predicted to be 22% (Fig. 4). The efficacies for benzene
(56%), 2-cyclopenten-1-one (6%), and 3-methyl-2cyclopenten-1-one (2%) were estimated based on the initial
dose-response curves for the ciliary beat frequency assay (Fig.
4). For the oocyte pickup rate assay, 5-methylindole (73%)
had a higher estimated efficacy than indole (52%) (Fig. 5). The
estimated efficacies of quinoline (70%) and isoquinoline
(61%) were high for oocyte pickup rate (Fig. 5). 3-Methyl-2cyclopenten-1-one (20%) had a lower efficacy in the oocyte
pickup rate assay than 2-cyclopenten-1-one (66%) (Fig. 5).
The efficacy of benzene in the oocyte pickup rate assay was
predicted to be 52% (Fig. 5). The exact efficacies in the
smooth muscle contraction rate assay could not be determined
based on the initial dose-response curves (Fig. 6). The
efficacies could only be estimated for: quinoline (75%),
2-cyclopenten-1-one (61%), isoquinoline (60%), indole
(55%), 3-methyl-2-cyclopenten-1-one (45%), 5-methylindole
(39%), and benzene (26%) (Fig. 6).
DISCUSSION
The purpose of this study was to identify additional
compounds in cigarette smoke that inhibit oviductal functioning. Phenol, 10 phenolic derivatives, indole, 5-methylindole,
148
RIVELES, ROZA, AND TALBOT
TABLE 4
Hierarchies of Potency in the Ciliary Beat Frequency,
Oocyte Pickup Rate, and Infundibular Smooth Muscle
Contraction Rate Assays
LOAELs
Rank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Name
CBF
OPRa
SMC
Indole
Isoquinoline
4-Ethylphenol
Quinoline
4-Methylphenol
2-Methylphenol
5-Methylindole
2,6-Dimethoxyphenol
Hydroquinone
3-Methyl-2-cylcopenten1-one
2,4-Dimethylphenol
2-Methoxyphenol
2-Cyclopenten-1-one
4-Methoxyphenol
2-Ethylphenol
2,5-Dimethylphenol
Benzene
Phenol
1013
1012
1011
1013
1012
109
1010
1010
1010
Xb
1014
1013
1012
1011
1011
1011
1011
1011
1010
1010
1015
1013
1012
1011
1011
1011
1010
109
1010
1010
109
108
107
107
105
108
108
101
1010
1010
109
108
108
107
107
102
109
108
109
107
107
106
106
102
a
Hierarchy of chemicals created based on LOAELs in the oocyte pickup rate
(OPR) assay.
b
‘‘X’’ means that the LOAEL could not be determined at the highest dose
tested for this assay.
quinoline, isoquinoline, benzene, 2-cyclopenten-1-one, and
3-methyl-2-cyclopenten-1-one were identified in fractions of
mainstream smoke solutions that inhibited oviductal functioning. The hierarchy of potency of the individual chemicals
tested in this study and their LOAELs in the ciliary beat
frequency, oocyte pickup rate, and infundibular smooth muscle
contraction rate assays are summarized in Table 4. Several of
these smoke toxicants were highly effective at femtomolar or
picomolar doses in all three oviductal assays (indole, isoquinoline, 4-ethylphenol, quinoline, 4-methylphenol, 5-methylindole,
and hydroquinone) (Table 4).
Previous studies have shown inhibition of oviductal functioning in picomolar or nanomolar ranges by pyridines (Riveles
et al., 2003) and pyrazines (Riveles et al., 2004). While
pyridine itself was not very potent in the oviductal assays,
addition of a single methyl or ethyl group increased its potency
greater than a 100-million-fold. Within the pyrazine group,
addition of single ethyl and methyl groups had minor effects on
its potency, in most cases. As was the case for the pyridines,
substitution of an ethyl or methyl group to the ring increased
the potency of the phenolic derivatives. In the case of the
phenols, this increase was clearly correlated with the position
of the substitution.
The LOAELS for phenol and its derivatives ranged from
1012 M to 101 M. Four phenolic compounds (2-methylphenol, 4-ethylphenol, 2,6-dimethoxyphenol, and hydroquinone)
had both high potencies and high efficacies in all three
oviductal assays. 2,4-Dimethylphenol was potent in picomolar
doses in all three assays, but only had high efficacies for the
ooycte pickup rate and smooth muscle contraction rate assays.
Four phenolic derivatives (2-ethylphenol, 2,5-dimethylphenol,
2-methoxyphenol, and 4-methoxyphenol) had high efficacies,
but medium or low potencies.
Addition of either a methyl or ethyl group to the two or
four position of phenol increased its potency in the oviductal
assays. In general, the four substitution resulted in higher
potency than the two. For the oocyte pickup rate and smooth
muscle contraction rate assays, substitution of an ethyl group
in the four position increased potency more than a two or
four methyl substitution. Substitution of a methyl group in
the two position or an ethyl group in the two or four
positions was also highly efficacious in all three assays. 4Methylphenol was very potent in all three oviductal assays,
but had very low efficacies. The parent compound, phenol,
was the least potent chemical in the oviductal assays with the
highest LOAELs (102 to 101 M). Phenol was very
effective in the oocyte pickup rate and smooth muscle
contraction rate assays, but only at very high doses. In
general, both substitution and position of the substitution
influenced the degree of toxicity and efficacy in the bioassays. The data in this study show that several phenolic
compounds are highly effective and inhibitory of oviductal
functioning at very low doses.
Although the mechanism of action of phenols on the oviduct
is not known, phenolic compounds studied previously exerted
toxicity through the formation of phenoxy-free radicals (Smith
et al., 2002). High concentrations of phenoxy radicals at the
oviduct could attack biochemical processes such as production
of ATP, cAMP, Ca2þ, or nitric oxide (NO), which are important
in oviductal muscle contraction and ciliary beat frequency
(Cometti et al., 2003; Lansley et al., 1992; Lindblom and
Hamberger, 1980). The biochemical affected by the phenoxy
radical would depend on its interactions within the cell or on
its surface (Selassie et al., 1998). The hydrophobicity of the
phenoxy free radicals would enable them to penetrate into the
hydrophobic region of the membranes (Selassie et al., 1998).
This could facilitate interaction with polyunsaturated fatty
acids and initiate free radical chain reactions (Selassie et al.,
1998). In our study, one possible mechanism by which the
phenolic compounds exerted toxicity on the oviduct may have
been through the formation of phenoxy radicals. The free
radicals produced and circulated to the oviduct may interfere
with the production of biochemicals necessary for adhesion of
the oocyte to the tips of the cilia on the infundibular surface.
These free radicals could also inhibit oviductal functioning by
slowing or stopping smooth muscle contraction by interfering
with ATP or NO production. The oxidative damage produced
SMOKE TOXICANTS INHIBIT OVIDUCT FUNCTION
by phenoxy free radicals could also result in cell death or
mutation (Selassie et al., 1998).
Previous studies have evaluated the reproductive toxicity of
phenol and its derivatives (Bian and Wang, 2004; Bruce et al.,
1987). Phenol itself is rapidly absorbed and distributed to all
tissues in animals and humans after oral, dermal, or inhalation
exposure and has a half-life of approximately 3.5 h in humans
(Bruce et al., 1987). Oral doses of phenol in rats showed doserelated fetal toxicity with a decrease in average fetal body
weight per litter (Bruce et al., 1987; Narotsky and Kavlock,
1995). Reduction of growth (decreased somite number) and
development (tail bud abnormalities) were observed in embryo
cultures in vitro after exposure to phenol and its parasubstituted cogeners (Oglesby et al., 1992). In vivo exposure
to phenol decreased weight gain in pregnant rats (Kavlock,
1990). A limited number of studies have shown that phenol and
its derivatives have reproductive effects (Bian and Wang, 2004)
and our study clearly shows that certain phenolic compounds
interfere with proper oviductal functioning in vitro in as low as
picomolar and nanomolar doses.
Phenolic compounds have been measured in cigarettes
(Chen and Moldoveanu, 2003; Czogala and Wardas, 1998;
Forehand et al., 2000; Nanni et al., 1990; Rustemeir et al.,
2002; White et al., 1990). The concentration of phenol and
its derivatives in cigarette smoke varies significantly among
different brands of cigarettes (Nanni et al., 1990). For example,
in most cases, commercial brands (3.6–17 lg/cigarette) had
higher concentrations of phenol than 1R4F research cigarettes
(7 lg/cigarette) or ultra-low-tar cigarettes (0.26 lg/cigarette)
(Nanni et al., 1990). However, the concentrations of phenol
vary among different research brands, for example, 1R3
cigarettes (55.4 lg/cigarette) have more phenol than 1R4F
cigarettes (6.9 lg/cigarette) (Forehand et al., 2000). In our
study, phenol was identified in the MS smoke solutions from
2R1 cigarettes and in the active fractions. Phenol was the least
potent chemical tested in this study with LOAELs in the
millimolar range; however, it was very effective at these high
doses. Phenol (5.7 lg/ml) and 4-methylphenol (1.78 lg/ml)
have been detected in the urine of smokers (Orejuela and Silva,
2002). Taken together, the data suggest that phenol, if present
in cigarette smoke in high enough concentration, could enter
the circulatory system and inhibit oviductal functioning,
especially oocyte pickup rate and smooth muscle contraction
rate, effectively.
In addition to phenol, the concentration of hydroquinone has
been measured in 1R3 research cigarettes (70.5 lg/cigarette)
(Forehand et al., 2000) and MS smoke from a nonfilter
cigarette (110–300 lg/cigarette) (U.S. EPA, 1992). In the
current study, hydroquinone was highly potent and efficacious
in all three oviductal assays. The concentration of hydroquinone in MS smoke could be sufficient to account for the effects
we observed in vitro on hamster oviductal functioning.
Humans are also exposed to phenols through foods (Bruce
et al., 1987). 2-Methoxyphenol, also known as guaiacol, is an
149
additive in cigarettes (http://www.rjrt.com/TI/TIcig_ingred_
summary.asp, Cigarette Ingredients, 2004), a Food and Drug
Administration (FDA) approved food additive, and is on the
Flavor and Extract Manufacturer’s Association (FEMA) generally regarded as safe (GRAS) list. It is also found in celery,
cocoa, coffee, rum, soybean, tea, tomato, whiskey, wine, and
used in ice cream, baked goods, meat, chewing gum, and dairy
products (http://www.bbc.co.uk/worldservice/sci_tech/features/
health/tobaccotrial/inacigarette599.htm, What’s in a Cigarette,
2004). In our study, 2-methoxyphenol inhibited oviductal
functioning in nanomolar and picomolar doses and was highly
efficacious in all three assays. 4-Ethylphenol and 2,6dimethoxyphenol are also on the FEMA GRAS list and are
found and used in a variety of consumer products. In the
current study, 4-ethylphenol and 2,6-dimethoxyphenol had
both high efficacies and high potencies in all three oviductal
assays.
The LOAELs for the indole, quinoline, 2-cyclopenten-1-one
compounds, and benzene ranged from 1015 M to 106 M.
Four compounds (indole, 5-methylindole, quinoline, and isoquinoline) had both high potencies (femtomolar and picomolar
doses) and high efficacies in all three oviductal assays. Benzene
had a high efficacy and medium potency (nanomolar doses) for
ciliary beat frequency and oocyte pickup rate, but a lower
potency (micromolar doses) for the smooth muscle contraction
rate assay. 2-Cyclopenten-1-one and 3-methyl-2-cyclopenten1-one were highly potent for the oocyte pickup rate and
contraction rate assays, but not for the ciliary beat frequency
assay, and they had varying efficacies.
Two of the most potent and efficacious cigarette smoke
components tested in this study were indole and 5-methylindole.
Although reproductive studies were not found on these two
compounds, another type of indole, indole-3-acetic acid (I3C), is
a negative regulator of estrogen (Auborn et al., 2003) and has
been shown to be teratogenic in mice and rats at 500 mg/kg/day
resulting in decreased weight gain (John et al., 1979). I3C is an
endocrine disrupter that can block ovulation in adult rats at 0.5
to 1.5 g/kg/day and cause decreases in body and ovarian weight
gain at 1.5 g/kg/day (Gao et al., 2002). In the current study,
ciliary beat frequency, oocyte pickup rate, and oviductal smooth
muscle contraction were inhibited by indole in femtomolar
doses and 5-methylindole in picomolar doses in vitro. The
results from our study combined with previous studies suggest
that indoles are effectors that can adversely affect several
reproductive processes including oviductal functioning.
Although information is available on phenols and certain
indoles, the reproductive effects of quinoline and isoquinoline
are unknown. In our study, both quinoline and isoquinoline
inhibited oviductal functioning and were highly efficacious at
very low doses. Quinoline has been measured in the MS smoke
from 1R4F (0.30 lg/cigarette) and 2R4F (0.23 lg/cigarette)
research cigarettes (Chen and Moldoveanu, 2003); however
smoke condensate from 1R4F cigarettes had lower levels of
quinoline (0.19 lg/cigarette) (White et al., 1990). Higher
150
RIVELES, ROZA, AND TALBOT
concentrations of quinoline were reported for a nonfiltered
cigarette (0.5–2 lg/cigarette) (U.S. EPA, 1992). In our study,
quinoline inhibited oviductal functioning at femtomolar or
picomolar concentrations. Therefore, even small concentrations of quinoline in cigarettes may adversely affect reproductive processes.
In the current study, benzene was inhibitory of oviductal
functioning at nanomolar or micromolar doses in the oviductal
assays and had efficacies greater than 50% for two of the assays. Smokers inhale greater concentrations of benzene (2 mg/day)
than nonsmokers (0.2 mg/day) as measured by breath levels
(Wallace et al., 1987). Commercial cigarettes contain, on average, more benzene (50 lg/cigarette) (Darrall et al., 1998) than
1R4F (44.33 lg/cigarette) and 2R4F (43.39 lg/cigarette) research cigarettes (Chen and Moldoveanu, 2003). Occupational
benzene exposure during pregnancy has been associated with
an increased incidence of spontaneous abortion (Marinova,
1978), which can occur as a result of diminished oviductal
functioning. Taken together, these results suggest that benzene
contributes to the inhibition of oviductal functioning observed
in hamster oviducts in vitro.
In summary, our study has shown that indole, 5-methylindole, quinoline, isoquinoline, 2-methylphenol, 4-ethylphenol, 2,6-dimethoxyphenol, and hydroquinone inhibit oviductal
functioning of hamsters in vitro at very low doses and high
efficacies. Based on the LOAELs determined in our study, it
is likely that the doses of these compounds received from
cigarette smoke inhalation are high enough in smokers to be of
concern to both pregnant and nonpregnant women. Exposure of
women to phenols, quinolines, and indoles through both
cigarette smoke and other sources may pose reproductive
problems. Our data demonstrate the need for further reproductive studies on these groups of compounds to determine if they
are linked to the increased incidence of ectopic pregnancy and
spontaneous abortion observed in women who smoke (Saraiya
et al., 1998).
ACKNOWLEDGMENT
This work was funded by grants 10RT-0239 and 13RT-0068 from the
Tobacco-Related Disease Research Program, California.
REFERENCES
Auborn, K. J., Fan, S., Rosen, E. M., Goodwin, L., Chandraskaren, A.,
Williams, D. E., Chen, D., and Carter, T. H. (2003). Indole-3-carbinol is
a negative regulator of estrogen. ASNS Suppl. (Nutritional Genomics in
Cancer Processes), 247OS–2475S.
Bian, Q., and Wang, X. (2004). Toxic effect and mechanism of alkyl-phenol
compounds on reproduction and development. Wei Sheng Yan Jiu. 33,
357–360.
Bruce, R. M., Santodonato, J., and Neal, M. W. (1987). Summary review of the
health effects associated with phenol. Toxicol. Ind. Health. 3, 535–568.
Castles, A., Adams, E. K., Melvin, C. L., Kelsch, C., and Boulton, M. L. (1999).
Effects of smoking during pregnancy. Five meta-analyses. Am. J. Prev. Med.
16, 208–215.
Chen, P. X., and Moldoveanu, S. C. (2003). Mainstream smoke chemical
analyses for 2R4F Kentucky reference cigarette. Contrib. Tob. Res. 20,
448–458.
Cometti, B., Dubey, R. K., Imthurn, B., Jackson, E. K., and Rosselli, M. (2003).
Oviduct cells express the cAMP-adenosine pathway. Biol. Reprod. 69,
868–875.
Czogala, J., and Wardas, W. (1998). Content of volatile in steam phenols in
mainstream of cigarette smoke from selected brands of cigarettes. Rocz.
Panstw. Zakl. Hig. 49, 365 (abstract).
Darrall, K. G., Figgins, J. A., Brown, R. D., and Phillips, G. F. (1998).
Determination of benzene and associated volatile compounds in mainstream
cigarette smoke. Analyst 123, 1095–1101.
DiCarlantonio, G., and Talbot, P. (1999). Inhalation of mainstream and
sidestream cigarette smoke retards embryo transport and slows muscle
contraction in oviducts of hamsters (Mesocricetus auratus). Biol. Reprod. 61,
651–656.
Florek, E., and Marszalek, A. (1999). An experimental study of the influences
of tobacco smoke on fertility and reproduction. Hum. Exp. Toxicol. 18,
272–278.
Forehand, J. B., Dooly, G. L., and Moldoveanu, S. C. (2000). Analysis of
polycyclic aromatic hydrocarbons, phenols and aromatic amines in particulate phase cigarette smoke using simultaneous distillation and extraction as
a sole sample clean-up step. J. Chromatogr. A 898, 111–124.
Gao, X, Petroff, B. K., Oluola, O., Georg, G., Terranova, P. F., and Rozman,
K. K. (2002). Endocrine disruption by indole-3-carbinol and tamoxifen:
Blockage of ovulation. Toxicol. Appl. Pharmacol. 183, 179–188.
John, J. A., Blogg, C. D., Murray, F. J., Schwetz, B. A., and Gehring, P. J.
(1979). Teratogenic effects of the plant hormone indole-3-acetic acid in mice
and rats. Teratology 19, 321–324.
Kavlock, R. J. (1990). Structure-activity relationships in the developmental
toxicity of substituted phenols: In vivo effects. Teratology 41, 43–59.
Knoll, M., Shaoulian, R., Magers, T., and Talbot, P. (1995). Ciliary beat
frequency of hamster oviducts is decreased in vitro by exposure to solutions
of mainstream and sidestream cigarette smoke. Biol. Reprod. 53, 29–37.
Knoll, M., and Talbot, P. (1998). Cigarette smoke inhibits oocyte cumulus
complex pick-up by the oviduct independent of ciliary beat frequency.
Reprod. Toxicol. 12, 57–68.
Lansley, A. B., Sanderson, M. J., and Dirksen, E. R. (1992). Control of the beat
cycle of respiratory tract cilia by Ca2þ and cAMP. Am. J. Physiol. 263,
L232–L242.
Lindblom, B., and Hamberger, L. (1980). Cyclic AMP and contractility of the
human oviduct. Biol. Reprod. 22, 173–178.
Magers, T., Talbot, P., DiCarlantonio, G., Knoll, M., Demers, D., Tsai, I., and
Hoodbhoy, T. (1995). Cigarette smoke inhalation affects the reproductive
system of female hamsters. Reprod. Toxicol. 9, 513–525.
Marinova, G. (1978). Problems of interrupted pregnancy among working
women. Akush Ginekol 17, 412 (abstract) .
Mitchell, J. H., and Hammer, R. E. (1985). Effects of nicotine on oviducal
blood flow and embryo development in the rat. J. Reprod. Fertil. 74, 71–76.
Mueller, L., and Ciervo, C. A. (1998). Smoking in women. J. Am. Osteopath.
Assoc. 98 (Suppl.), S7–S10.
Nanni, E. J., Lovette, M. E., Hicks, R. D., Fowler, K. W., and Borgerding, M. F.
(1990). Separation and quantitation of phenolic compounds in mainstream
cigarette smoke by capillary gas chromatography with mass spectrometry in
the selected-ion mode. J. Chromatogr. 505, 365–374.
SMOKE TOXICANTS INHIBIT OVIDUCT FUNCTION
Narotsky, M. G., and Kavlock, R. J. (1995). A multidisciplinary approach to
toxicological screening: II. Developmental toxicity. J. Toxicol. Environ.
Health 45, 145–171.
Neri, A., and Eckerling, B. (1969). Influence of smoking and adrenaline
(epinephrine) on the uterotubal insufflation test (Rubin test). Fertil. Steril. 20,
818–828.
Neri, A., and Marcus, S. L. (1972). Effect of nicotine on the motility of the
oviducts in the Rhesus monkey: A preliminary report. J. Reprod. Fertil. 31,
91–97.
Oglesby, L. A., Ebron-McCoy, M. T., Logsdon, T. R., Copeland, F., Beyer, P. E.,
and Kavlock, R. J. (1992). In vitro embryotoxicity of a series of parasubstituted phenols: Structure, activity, and correlation with in vivo data.
Teratology 45, 11–33.
Orejuela, E., and Silva, M. (2002). Sensitive determination of low molecular
mass phenols by liquid chromatography with chemiluminescence detection
for the determination of phenol and 4-methylphenol in urine. Analyst 127,
1433–1439.
Riveles, K., Iv, M., Arey, J., and Talbot, P. (2003). Pyridines in cigarette smoke
inhibit hamster oviductal functioning in picomolar doses. Reprod. Toxicol.
17, 191–202.
Riveles, K., Roza, R., Arey, J., and Talbot, P. (2004). Pyrazine derivatives
in cigarette smoke inhibit hamster oviductal functioning. Reprod. Biol
Endocrinol. 2, 23–27.
Ruckebusch, Y. (1975). Relationship between the electrical activity of the
oviduct and the uterus of the rabbit in vivo. J. Reprod. Fertil. 45, 73–82.
Rustemeir, K. R., Stabbert, R., Haussmann, H. J., Roemer, E., and Carmines,
E. L. (2002). Evaluation of the potential effects of ingredients added to
151
cigarettes. Part 2: Chemical composition of mainstream smoke. Food Chem.
Toxicol. 40, 93–104.
Saraiya, M., Berg, C. J., Kendrick, J. S., Strauss, L. T., Atrach, H. K., and
Ahn, Y. W. (1998). Cigarette smoking as a risk factor for ectopic pregnancy.
Am. J. Obstet. Gynecol. 178, 493–498.
Selassie, C. D., Tushini, D., DeSoyza, V., Rosario, M., Gao, H., and Hansch, C.
(1998). Phenol toxicity in leukemia cells: A radical process? Chem. Biol.
Interact. 113, 175–190.
Shiverick, K. T., and Salafia, C. (1999). Cigarette smoking and pregnancy I:
Ovarian, uterine and placental effects. Placenta 20, 265–272.
Smith, C. J., Perfetti, T. A., Morton, M. J., Rodgman, A. Garg, R., Selassie,
C. D, and Corwin, H. (2002). The relative toxicity of substituted phenols
reported in cigarette mainstream smoke. Toxicol. Sci. 69, 265–278.
Talbot, P., DiCarlantonio, G., and Knoll, M. (1998). Identification of cigarette
smoke components that alter functioning of hamster (Mesocricetus auratus)
oviducts in vitro. Biol. Reprod. 58, 1047–1053.
U.S. EPA (1992). EPA Report/600/6–90/006F: Respiratory health effects of
passive smoking: Lung cancer and other disorders. U.S. Environmental
Protection Agency, Washington, DC.
Wallace, L., Pellizzari, E., Hartwell, T. D., Perritt, R., and Ziegenfus, R. (1987).
Exposures to benzene and other volatile compounds from active and passive
smoking. Arch. Environ. Health 42, 272–279.
White, E. L., Uhrig, M. S., Johnson, T. J., Gordon, B. M., Hicks, R. D.,
Borgerding, M. F., Coleman, W. M., and Elder, J. F. (1990). Quantitative
determination of selected compounds in a Kentucky 1R4F reference
cigarette smoke by multidimensional gas chromatography and selected ion
monitoring-mass spectrometry. J. Chromatogr. Sci. 28, 393–399.