Abstract
Atrazine, a s-triazine herbicide, is the most widely used herbicide in the U.S. and
is also used in over 80 countries. It is of concern since after spring rains and by leeching
processes, atrazine and its breakdown products (considered equally as toxic) enter water
supplies, streams, and other major bodies of water. It reaches levels in community water
systems up to 89 ppb, in rivers basins up to 131 ppb, in drinking water up to 12 ppb, and
even in rainfall up to 40 ppb. Atrazine alters neuroendocrine function, causes mammary
tumors in rats, and a recent study showed demasculation and hermaphroditism in frogs at
concentrations as low as 1 ppb.
This study determined whether atrazine could compromise the multixenobiotic
resistance (MXR) mechanism which is a mechanism for preventing toxicants from
entering cells. I found that atrazine affects the normal functioning of MXR in the gill
tissue of the mussel Mytilus californianus down to a concentration of 0.6 uM (127 ppb).
Atrazine’s effect on development was also examined and was found to have no effect on
the development of the sea urchin, Lytechinus pictus at a concentration of 2 uM (423
ppm).
The high levels of atrazine in aquatic environments due to agricultural runoff,
coupled with the significant effects of atrazine on the MXR mechanism raises concern
over the health of other marine organisms. Atrazine's inhibition of the MXR substrate
potentially could allow other toxic or carcinogenic agents to enter cells and adversely
affect the health of marine communities.
Introduction
Atrazine is a s-triazine herbicide that has been used for over 40 years to kill broad
leaf and grassy weeds by inhibition of photosynthesis [fig 1). It is used in over 80
countries (Hayes et al., 2002), and in the U.S. alone accounts for 86% of herbicides. The
U.S., primarily the Midwest, uses 76.4 million pounds per year. Atrazine is applied to
76% of the corn grown in the U.S. as well as to sorghum, sugarcane, wheat, guava,
macadamia nuts, hay, summer fallow, forestry, Christmas trees, sod, and residential turf
(parks, golf courses, private lawns).
Given the large amount of atrazine in the environment, toxicological studies on
atrazine are very important. The LD5O in rats is 1.75 ppm (Islam, 2001). However, there
have been reported effects at concentrations which are approximately 1000 times lower.
Atrazine has been reported by the EPA to cause neuroendocrine effects in rats at
concentrations of 18 ppb. The most recent and perhaps most striking study found that
concentrations of atrazine down to 0.1 ppb induced hermaphroditism and demasculation
of the larynges of male African clawed frogs, Xenopus laevis (Hayes et al., 2002). At
concentrations as low as 25 ppb, there was a tenfold decrease in testosterone in these
frogs. It was proposed that atrazine decreased testosterone production and increased
estrogen production.
These toxicological studies have been limited and targeted specific systems such
as the endocrine system. This study investigates the toxicity of atrazine on a more general
cellular mechanism. This mechanism is the multixenobiotic resistance (MXR)
mechanism and is one of two ways in which cells deal with toxicants. In the first
mechanism the toxicant enters the cell and is broken down or converted to other
compounds through the cytochrome P450 system (Livingstone, 1990) or conjugating
system which makes the toxicant more water soluble (Ortiz de Montellano, 1986). A
problem with this system is that the toxicant must enter the cell and it could damage the
cell before the toxicant is altered.
The second mechanism of detoxification is the MXR mechanism, first identified
in human tumor cells, which actively exports moderately hydrophobic chemicals which
diffused into the cell of cell plasma membrane (Dano, 1973; Gottesman et al., 1991.)
This transport occurred through an ATP-dependent, transmembrane, 170 kDa p-
glycoprotein. These proteins are part of a larger family of proteins, named the ABC
proteins, which are generally specified for transport (Epel, 1998).
The p-glycoproteins active in the MXR mechanism have been identified in human
tissue in the kidney, adrenal gland, liver, the blood-testes barrier (Cordon-Cardo et al.,
1990), and the blood brain barrier (Watchko et al., 1998). Since then, a related 170 kDa
p-glycoprotein has been detected and identified in many other organisms by genetics, by
activity measurements, and by immunological probes. The MXR mechanism is present in
many aquatic organisms such as fresh water and marine mussels including Mytilus
californianus (Cornwall et al., 1995). Öther marine organisms with MXR are sponges
(Kurelec et al., 1992), snails (Kurelec et al., 1995a), clams (Waldmann et al., 1995) and
worms (Toomey and Epel, 1993). The MXR mechanism has been suggested as a first line
of defense against xenobiotics in aquatic environments (Epel, 1998).
An assay using the accumulation of a radiolabeled or fluorescent substance was
designed in order to detect MXR activity (Kurelec et al., 1992; Toomey and Epel, 1993;
Kurelec et al., 2000). Rhodamine B is a fluorescent dye which normally only
accumulates in cells at low levels as it is a MXR substrate and is constantly effluxed from
the cell. However, when rhodamine B is put into solution with a chemical which are
substrates or inhibitors of the p-glycoprotein, rhodamine B accumulates in the cell at
much higher levels; the MXR mechanism is basically overwhelmed by such multiple
substrates or inhibitors. Therefore, even if a chemical itself may not cause adverse
cellular effects, as an MXR substrate it would allow other more hazardous toxicants to
enter the cells.
I chose to determine whether atrazine was an MXR substrate for several reasons:
atrazine is hydrophobic, the end destination of much agricultural run-off is the ocean, and
for many sessile marine organisms, the MXR mechanism may be their last line of defense
against toxicants. I also looked at the effect of atrazine on the development of embryos of
the sea urchin, Lytechinus pictus.
Atrazine was found to be an MXR substrate and to significantly inhibit MXR at
concentrations as low as 0.6 uM. There were no observed developmental effects on
Lytechinus pictus. This study raises the concern that even if atrazine itself is present at
non-toxic doses, its inhibition of MXR could make cells more vulnerable to other
toxicants which normally would not enter the cells.
Materials and Methods
Materials
Organisms.
Adult mussels of the species Mytilus Californianus were collected from the
exposed rocky intertidal shore at Hopkins Marine Station in Monterey, California. The
average shell length of the mussels collected was 7.5 cm. The mussels were kept outdoors
in plastic containers with fresh, filtered continuously flowing seawater at approximately
15 °C. The mussels were used within six days of collection. Adult specimens of
Lytechinus pictus from Marinus, Venice, California were kept in glass tanks outdoors
with fresh, flowing seawater at approximately 15%
Chemicals.
Atrazine was ordered from Chemical Service Inc. (West Chester, PA). Stock
solutions of 10 mM, SmM, and 1mM were made by diluting Atrazine in DMSO. Stock
solutions were stored in plastic vials at room temperature. Rhodamine B was ordered
from Sigma. A ImM stock solution was made by diluting Rhodamine B in deionized
water. Verapamil was ordered from Sigma, and a 20 mM stock solution was made by
diluting verapamil in DMSO. Both the rhodamine B and verapamil stocks were kept in
15 ml polypropylene conical tubes covered in foil at room temperature.
Methods
The methods used for mussel gill preparation and the rhodamine accumulation assay
were modified from procedures described by Chan, 2000; Eufemia and Epel, 2000; and
Cornwall et al., 1995. The procedure with modifications is described below.
Preparation of Gill Tissue.
Gill tissue was removed intact from freshly dissected Mytilus californianus and
placed in a petri dish with fresh seawater at approximately 15 °C. Excess mucus was
removed from the intact gills with forceps. The gills were then cut into approximately
Amm x 4mm pieces. Effort was made to have the gill pieces be only one tissue layer thick
by teasing apart with forceps the ascending and descending arms of the gill tissue
(Cornwall et al., 1995). The 4mm x 4mm pieces of tissue were incubated at 15 °C for 15
minutes in a separate petri dish with fresh seawater in order to let excess mucus flush out.
At the end of the 15 minute incubation, excess clumps of mucus were removed again
from the pieces of tissue with forceps. All gill tissue was used for experimentation within
one hour of dissection.
Rhodamine Accumulation in Gill Tissue Over Time.
While the gill tissue pieces were incubating in seawater (as above) to remove
mucus, 35 x 10 mm petri dishes were set up with five mL of fresh seawater each and the
following substrate or inhibitor concentrations: 0.6 uM Rhodamine B/ SuM verapamil;
0.6 uM Rhodamine B/ SuM Atrazine; and 0.6 uM Rhodamine B only. Preliminary
experiments showed that a 0.6 uM rhodamine concentration was in the range to detect
efflux activity accurately.
Six to seven pieces of gill tissue were placed in each petri dish and incubated at
15 °C. After intervals of 30, 45, and 60 minutes, one or two pieces of gill tissue were
removed from each solution. The tissues were immediately washed with fresh seawater
by immersion in 300 mL of seawater for 15 seconds and then laid flat on a new petri dish
with no water except the moisture that came with the tissue itself. Tissues were viewed
under the microscope within five minutes of removal from solution.
The fluorescence intensity of the tissue samples was viewed and measured using
an epifluorescent microscope with a rhodamine filter and a 16X objective. A camera
(Hamamatsu, model C2400-08) was linked to the microscope as well as a PC with a
Universal lmage processing system (Image-1) for analysis of data. The sensitivity and
intensity of the light on the microscope was set at the beginning of the experiment and
left untouched for the rest of the experiment in order to insure consistency of results. For
each piece of gill tissue, five fluorescence intensities of a 150-pixil area were recorded. In
addition, three background intensities were recorded. The methods for averaging these
recordings are described below in the statistics section.
Rhodamine Accumulation Concentration Series for 1 Hour
While previously dissected and cut gill tissue pieces incubated at 15 °C, 35 x 10
mm petri dishes with five mL fresh seawater were set up with the following
concentrations: 0.6 uM Rhodamine B/20 uM verapamil; 0.6 uM Rhodamine B/2 uM
DMSO; 0.6 uM Rhodamine B only, and 0.6uM Rhodamine B with various
concentrations of atrazine. DMSO controls were set up for threé of the four mussels
examined.
Five to six pieces of gill tissue were placed in each petri dish at one concentration
and incubated at 15 °C. The incubations were staggered with a ten minute interval
between the initiation of each incubation at each concentration. After 1 hour the pieces of
tissue were removed and immediately washed for 15 seconds in fresh seawater. The
tissue pieces were laid flat on new petri dishes and viewed under the microscope. The
relative intensity of a 100-pixil area was determined for each tissue.
Developmental Effects on Lytechinus pictus
One female and one male Lytechinus pictus were spawned. The eggs were
fertilized and allowed to develop in 2 uM, .5 uM, and .1 uM solutions in 30 mL beakers.
There were two beakers of each concentration, as well as a DMSO control and two
seawater controls. The beakers were covered in parafilm and allowed to develop at room
temperature. The embryos were examined and compared to controls are various time
points. The skeleton morphology was studied by lysing five day old embryos in a 2.5%
SDS (in dionized water) solution, and then skeletons were examined microscopically at
160x and 400x magnifications.
Statistics
For both the rhodamine accumulation over time as well as the concentration
series, the same procedure was used to average the intensity recordings. The five
intensity readings for each tissue were averaged as were the three background readings.
The average background reading was subtracted from the average tissue fluorescence
reading. The readings with background adjustment were then averaged across the five
tissue samples to produce a mean, final fluorescence intensity at that concentration. The
differences between these means and their respective controls were determined using the
Student's t-test to a significance of P-0.01.
Results
Kinetics of Rhodamine Accumulation in Gill Tissue
The kinetics of rhodamine accumulation in the presence and absence of atrazine
or verapamil is shown in Figure 4. At all three time points of 30, 45 and 60 minutes, the
rhodamine accumulation in the atrazine treated solutions were well above the control and
approaching the levels seen with verapamil. Similar results were seen with 4 uM atrazine
(results not shown). As seen in Figure 4, there are larger standard deviations at 60
minutes, which I attribute to mucus accumulation with resultant high fluorescence. This
variation (and excess mucus) were not seen later in the experiments (fig. 5-8) as my skills
at removing mucus improved.
The Effects of Different Concentrations of Atrazine
In the next set of experiments, time was a constant, and tissue samples were taken
at one hour since the above kinetics experiment indicated a maximum concentration was
reached after one hour. This procedure allowed for a comparison of different atrazine
concentrations in order to determine the lowest concentration at which atrazine would
significantly inhibit MXR.
A range of atrazine concentrations from 12 uM to 0.6 uM were used. As seen in
Figure 5, atrazine affects the MXR at all concentrations; even 0.6 uM was significantly
different than the Rhodamine control.
Since the series in Figure 5 showed significant effects of atrazine at 0.6 uM, a
new series was done at 0.6 uM and below to ascertain the minimal level where atrazine
had an effect. As seen in Figure 6, 0.6 uM atrazine was not different from the control at a
significance level of p=0.01 (only p-0.05), and there was no difference at 0.1 and 0.01
atrazine concentrations. There was no effect of DMSO.
The next series attempted to find the effect of atrazine at concentrations between
0.4 uM and 0.1 uM in order to narrow down the range of MXR inhibition.
Concentrations of 0.4 uM, 0.2 uM, and the DMSO control were all similar compared to
the rhodamine control [fig. 71.
The final series, shown in Figure 8, duplicated some of the concentrations from
previous experiments. The atrazine concentration of 0.6 uM had an effect significant to
p-0.01, but differences between the 0.5 uM, 0.4 uM as well as the DMSO control were
not significant compared to the rhodamine control [fig. 81.
Because there were variations in the results obtained from each individual mussel
[fig. 5-8), the data was normalized. For a given mussel, the verapamil fluorescence was
assumed to represent the fluorescence which would occur given no MXR activity. This
assumption was valid, as verapamil is known to completely inhibit p-glycoprotein
activity. The fluorescence of each concentration including the rhodamine control was
divided by the fluorescence of the verapamil concentration. The resulting percentages
represented the percentage of the verapamil concentration fluorescence, or in other
words, the percent of MXR inhibition. Two of the concentrations had more than one
percentage value because it had been repeated more than once in the trials in Figures 5-8,
and these percentages were averaged and a standard error calculated [fig. 9. As seen, the
0.6 uM atrazine concentration resulted in 44.6% of the rhodamine fluorescence seen with
verapamil. The differences between the 0.5 uM and 0.4 uM atrazine concentrations and
the rhodamine control were determined to be insignificant with a Student’s two-tailed t-
test. Therefore, this composite data confirms that atrazine significantly acts as an MXR
substrate down to 0.6 uM.
DMSO controls were used in most experiments in order to insure that any
differences in rhodamine fluorescence were attributable to atrazine and not the DMSO in
which it was dissolved. AIl DMSO controls in this experiment were not significant
compared to the rhodamine-only controls.
Developmental Effects on Lytechinus pictus
There were no clear timing or morphological differences in the Lytechinus pictus
embryos exposed to various concentrations of atrazine (0.1-2 uM). The spicule skeletons
of the embryos were examined after three days for any differences attributable to
atrazine. There were some skeletal defects, but they occurred in both the seawater
control, DMSO control, and at all atrazine concentrations; therefore, the defects could not
be attributed to atrazine or DMSO. In both the controls [fig. 21 and experimentals,
observed defects included overly curved larger legs and curved portions of the smaller
legs not coming together [fig. 3]. However, there were no apparent developmental effects
attributable to atrazine (2uM) on Lytechinus pictus embryos.
Discussion
This study shows that atrazine is an MXR substrate and significantly inhibits
multixenobiotic resistance down to a concentration of 0.6 uM. At this concentration,
atrazine allowed approximately 24% more rhodamine into the cell than would normally
enter with no competitive substrate. However, atrazine does not appear to affect the
development of Lytechinus pictus embryos at 2 uM.
Although each experiment assessing the effects of atrazine on mussels indicated
that atrazine was a significant substrate, there were significant differences in the levels of
rhodamine accumulation between the different experiments (different animals). These
differences in individual specimens have been observed in other studies, and have been
attributed to the differing levels of p-glycoprotein. The MXR response in Mytilus
californianus is induced (increased p-glycoprotein levels and efflux activity) in response
to environmental stresses (Eufemia and Epel, 2000). With lower levels of p-glycoprotein
activity, cells would naturally allow more rhodamine to accumulate than would cells with
high levels of p-glycoprotein. This relationship was expressed by Smital et al. (2000) as
an r-ratio. This ratio is the rhodamine control fluorescence divided by the verapamil
fluorescence in a given mussel. R-ratios ranged from zero to one, with numbers close to
one having very little p-glycoprotein activity, and levels close to zero having very high p-
glycoprotein activity.
Variations in p-glycoprotein efflux activity could explain the differences in the
basal fluorescence seen in the controls of the various mussels. The R-ratios were
determined for the mussels used in Figures 5-8 and were 0.281, 0.302, 0.130 and 0.146
respectively. The R-ratios of mussels A and B [fig. 5-6) were significantly different to a
0.01 level from the r-ratios of mussels C and D. The average of the r-ratios of A and B
was 2.12X the average of r-ratios of C and D [fig. 7-8). In order to determine whether
this 2.12-fold difference was consistent for all concentrations, the ratios of other
concentrations were determined. This difference was seen; the average of the
fluorescence of the 0.6 uM atrazine concentration for mussel A and B was 2.1 5X the 0.6
uM concentration for mussel D (no value was available for mussel C).
The higher R-ratios in mussels A and B imply a reduced level of MXR activity
compared to mussels C and D. This explains why the control rhodamine fluorescence for
mussels A and B was higher than it was in mussels C and D at similar test-
concentrations. Therefore, taking into consideration the differences in p-glycoprotein
activity between the mussels, most of the variation between results appears to be
attributable to the innate difference in MXR activity in these individual mussels, rather
than procedural errors. Most importantly, atrazine had the same effect on these different
groups.
The lowest concentration at which atrazine showed significant effects on MXR
activity in Mytilus californianus was 0.6 uM or 127 ppb; is this level environmentally
significant? A concentration of 127 ppb is of concern when considering the most recent
EPA report on atrazine risk assessment (EPA, 2002). In an assessment of community
water systems (CWS) in areas of atrazine use, 29 CWS had levels of contamination
higher than 12.5 ppb, and one was recorded at 89 ppb. The maximum level of
contamination in rural wells examined was 89 ppb, and eight of 1505 wells examined had
levels over 12.5 ppb (EPA, 2002). Among streams analyzed in Ohio, 13 flowing into
Lake Erie had concentrations ranging from 0.1 to 23.2 ppb. In the North Ohio Sandusky
river basin, eight sites had concentrations of atrazine ranging from 1-45.7 ppb. In the
Cape Fear River basin, North Carolina, 35% of the sites tested positive for atrazine with
an average of 5.6 ppm and a maximum of 131 ppb. In lowa concentrations of atrazine in
drinking water, rivers, water wells and reservoirs ranged up to 12 ppb. In a survey of
agricultural watersheds in southern Ontario in 1975-1977, 80% of the stream waters had
1.4 ppb atrazine (Spectrum Laboratories, 2001). That was 25 years ago. Atrazine is even
detected in 40 ppb in rainfall in some areas of the Midwest (Hayes, et al., 2002).
Clearly these water concentrations of atrazine are of concern, but when taking
into consideration the fact that atrazine may bioconcentrate (up to 11-fold, Spectrum
Laboratories, 2001) or have effects due to exposure over long periods of time, the
situation becomes of even greater concern. Indeed, the EPA itself this year admitted that
neuoroendocrine effects observed in rats due to atrazine are "not unlikely" in humans
(EPA, April 2002). In addition, natural fluctuations of p-glycoprotein expression raise
concerns about the timing of exposure to toxicants like atrazine. P-glycoprotein
expression is reduced during the embryonic and newborn period of rats, and reaches adult
levels only after 21 days of maturation (Tsai, et al. 2002).
However, because atrazine’s end destination is the aquatic environment, work is
also needed to investigate its effects in the marine realm. Specifically, research is needed
to determine concentrations of atrazine in marine waters, and whether marine organisms
are being affected. In addition, investigations are needed to determine whether other
herbicides or chemicals which are washed into marine waters are having similar adverse
effects of MXR activity or other biological functions.
Despite the numerous questions that still need to be answered regarding atrazine,
the work reported in this study adds a new dimension to understanding this herbicide.
Atrazine is not just a problem for humans or frogs, but is now identified as a possible
problem for aquatic organisms. Atrazine is a substrate for the MXR mechanism down to
levels of 127 ppb. Therefore, even if atrazine is present at low levels where it itself is not
toxic, its inhibition of MXR could make organisms more susceptible to other toxicants
that normally would be kept out of the cells.
Acknowledgments
I want to thank David Epel for inspiring this project, providing constant support
and suggestions as I reached apparent dead ends, and putting up with my mathematically-
challenged nature; Chris Patton for his much needed technical assistance; Becky, Todd,
and Cathy for including me into the lab, answering my questions, and making jokes; and
finally the many sea urchins and mussels which gave their lives for this project.
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Table 1: Student t-tests for Concentration Series Data In Question
Mussel
Concentration
Standard
t-value
Degrees of
Tested
deviation
freedom
of control
5.61
6 UM
10.53
8.12
DMSO
-1.26
3.7.
OI UM
8.12
DMSO
6.47
0.588
2 UM
6.47
2.11
DMSO
-3.98
1.80
1.80
-13.4
4UM
1.80
5 UM
1.80
6 UM
12.2
Significance
at 0.01
yes
yes
Legend
Fig. 1. Atrazine chemical structure.
Fig. 2. A normal skeleton of Lytechinus pictus at 6 days of development.
Fig. 3. An abnormal skeleton of Lytechinus pictus at 6 days of development.
Fig 4. The effect of 5 uM atrazine on the kinetics of xenobiotic transport. Error bars are
standard error of the mean of 5 tissues at each time point.
Fig 5. The effect of different atrazine concentrations on rhodamine accumulation (mussel
A). Error bars are the standard error of the mean of five tissue samples per sample.
Significance (*) is determined to p-0.01.
Fig 6. The effect of different atrazine concentrations on rhodamine accumulation (mussel
B). Error bars are the standard error of the mean of five tissue samples per concentration.
Fig 7. The effect of different atrazine concentrations on rhodamine accumulation (mussel
C). Error bars are the standard error of the mean of five tissue sample per concentration.
Fig 8: The effect of different atrazine concentrations on rhodamine accumulation (mussel
D). Error bars are the standard error of the mean of five tissue sample per concentration.
Significance (*) is determined to p=0.01.
Fig 9: The combined, normalized data of mussels from Figures 5-8 (mussels A-D). The
alues are th
ercentage of fluorescence above the rhodamine control. Verapamil
fluorescence was considered to represent 100% inhibition of the MXR mechanism.
Figure 1
Figure 2
Figure 3
Figure 4
140
120
100
80
40
20


——Verapamil (5 uM)
- - Atrazine (5 uM)
——Rhodamine (6 uM)
20
30
50
40
time (min)
60
70
Figure 5
78.5571
62.035
58.96
Concentrations
48.55
22.11
Figure 6
94.1
Concentrations
23.82
28.38
Figure 7
100
60
75.16
15.84
13.9
11.44
Verapamil (20 Atrazine (.4 uM) Atrazine (.2 uM) DMSO control
UN)
Concentrations
9.74
Rhodamine
control
Figure 8
100
76.7
11.8
11.2
Concentrations
Figure 9 : The % of fluorescence of Verapamil Concentrations
100
75
44.6
concentration
18.5 16.9
20.15