Effect of Herbicides on Blood Cell Progenitors
Larissa Ho, Selina Chin, and Prem Bassi
Biology/Mrs. Lemus
January 28, 2013
Abstract:
This experiment was conducted to determine the harmful effects of glyphosate on the production
of red blood cell colony formation. Colony formation was first started with dog blood
progenitors and growth factors. This was tested against a 1x chronic and acute exposure, as well
as a 5x chronic and acute exposure to the common herbicide Round-Up. Over the course of the
experiment, the results showed that there was a steady drop in the number and size of the
colonies formed by the dog blood cell progenitors. This shows that there is a negative effect of a
dog being exposed to glyphosate.
Research Question:
Does acute or chronic exposure to a common house-hold pesticide affect red blood cell
production? How do different concentrations of herbicides affect red blood cell progenitor
colony formation?
Background:
Pesticides are a mixture of substances used to destroy a life cycle of any pests found in crops
(Effects of Pesticides). According to the article Effects of Pesticides, pesticides affect farmers
and their families because it increases the risk of them getting toxic levels in their bodies. This is
because pesticides are carried on the wind, can be left on produce, and run off into the open
public water supply to humans, fish, and other seafood. The risk pesticides have on wildlife
depends on the type of pesticide, the persistence of the pesticide, and the ability to concentrate in
the wildlife food chain (Wildlife and Pesticides: A practical guide to reducing the risk). There are
many ways pesticides are exposed to wildlife. For example, wildlife is exposed to pesticides
when breathing them in, eating or drinking water contaminated with pesticides or by skin
absorption (Wildlife and Pesticides: A practical guide to reducing the risk). This can also be seen
as a cycle; birds mostly rely on insects for a food source. When insects are exposed to the
pesticides then the birds will also be exposed when they eat the insects.
Pesticides affect cells by deregulating the cell cycle (Glyphosate-based pesticides affect cell
cycle regulation). The cell first enters the cycle and goes into interface during the interface the
cell grows and prepares for mitosis which is where the cells divide. During interface the cells
grow by replicating DNA and they store food and energy for mitosis. Once the cell enters mitosis,
the cells divide to make somatic cells which are any cells in the body that aren’t germs. After the
cells divide they can carry out their normal cell functions or enter the cell cycle again and divide.
There have been many science studies done on dogs. They use animals to investigate human
affections, and used to see how the body works on drugs and product testing (Animal Testing).
Doctors usually draw blood samples and do test to determine the problem from humans, dogs,
cats, birds, and ferrets (Blood Cells & Complete Blood Counts (CBC) in Animals). A red count
is measuring the number of cells in the blood and the average range for most dogs is 8.7 x 106
RBC’s per microliter (Blood Cells & Complete Blood Counts (CBC) in Animals).
Hypothesis:
When the concentration of herbicides exposed to the blood cell progenitors increase, less red
blood cell colonies will form, and the size of colonies will decrease because according to studies
the deregulation of the cell cycle is an effect of being exposed to herbicides. What this means to
the cells is those herbicides affect the cell’s ability to divide and leads the cells into quiescence
and senescence. This means the cells will either go into hibernation or will get ready to die.
Independent Variables:
-Concentration of herbicides
-Acute Exposure or Chronic Exposure
Dependent Variable:
The number of colonies formed.
Constants:
Amount of cells in each well and the amount of time the cells are exposed to the herbicides.
Materials:
-Red Blood Cells from Dogs
-Centrifuge
-Sterile Tubes
-Incubator
-Pasteur Pipettes
-Herbicides
-Inverted Microscope
-Aspirator
-Well Plate
-Hemocytometer
Procedures:
1.
2.
3.
4.
5.
6.
Spin cells to get them out of the media using the centrifuge at 1500g’s for 5 minutes
Aspirate old media out of the tubes (only keep the pellet of cells)
Add 1 mL of new media then rack cells to mix in the new media
Add growth factors to promote cell growth (only for non-leukemic cells)
Bring total volume of solution to 10mL (using more media)
For acute exposure:
a. In two 15mL conical tubes add 0.5mL of cell suspension
b. Add 17ul of working pesticide solution (1x treatment)
c. Bring volume to 10mL by adding more media
d. Spin cells in a centrifuge for 5 minutes at 1500g’s
e. Aspirate the excess media off
f. Re-suspend total volume to 10mL
g. Aliquot 1mL in each well
h. Repeat steps a-g for 5x treatment, but add 85ul instead of 17ul
7. For chronic exposure:
a. In two 15mL conical tubes add 0.5mL of cell suspension
b. Add 17ul of working pesticide solution (1x treatment)
c. Bring volume to 10mL by adding more media
d. Aliquot 1mL in each well
e. Repeat steps a-d for 5x treatment, but add 85ul instead of 17ul
Data Collection and Processing:
Table 1. Colony Growth of Normal Dog Cells Exposed to Herbicides
Group
Dose
Acute Exposure
Chronic Exposure
(Micromolar)
Control
0
25
45
1x
0.2
15
30
5x
1
11
18
The data table above shows the number of colonies formed for each dose and exposure.
Sample Calculation:
During the experiment calculations were done in order to determine the number of cells in a
certain volume. Below is an example of a calculation that was done in order to determine the
number of cells.
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠
100
230
100
= # 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 × 1006
= 2.3 × 1006
= 2,300,000 cells
Table 2. Pictures of Colonies Formed
Picture
Description
A colony formed by the control group taken at
a low power (10x) on a microscope.
Two colonies formed by the 5x group taken at
a low power (10x) on a microscope.
An image of a colony from the control group
taken at high power (40x) on a microscope.
Figure 1. Colony growth of Normal Dog Cells
Exposed to Herbicides
# of Colonies Formed
50
40
Acute
30
y = -23.571x + 40.429
Chronic
20
y = -11.429x + 21.571
10
0
0
0.2
0.4
0.6
0.8
1
Herbicides Concentration
Figure 1 shows the colony growth for the specific exposure and the dosage of the herbicides. In
this figure, the blue points represent the acute exposure and the red points represent the chronic
exposure.
Conclusion:
This experiment did support the hypothesis for the lab because as the concentration of herbicides
increased the number of colonies formed decreased. According to Table 1, the control group for
the acute exposure had a number of 25 colonies formed and that decreased to 11 colonies when
the concentration of herbicides exposed increased to one micromolar. A similar trend happened
with the chronic exposure. The control group had 45 colonies formed and the group exposed to
one micromolar of herbicides had 18 colonies formed. Additionally, Figure 1 shows the
decreased in a visual form. As seen in the graph both the best fit lines for the acute and chronic
exposure are decreasing. This shows visually that as the herbicides concentration increased then
the number of colonies formed will decrease.
Evaluation:
In our methods there were a lot of potential errors that could have occurred. Our method of
counting was using a counter to record all the cells we saw. Some ways to improve this could
have been to have a machine count the cells, but we needed to practice with the skill of counting
and calculating the numbers. In the future we could have a dual microscope and have two people
count the same hemocytometer and confirm they have roughly the same number. To further
carry on this project we would like to test on leukemic cells.
Works Cited
Animal Testing: Dogs. Animal Port. November 16, 2012. Retrieved from
http://www.animalport.com/animal-testing/animal-testing-dogs.html
Blood Cells & Complete Blood Counts (CBC) in Animals, Pet Education.com. November 25,
2012. Retrieved from http://www.peteducation.com/article.cfm?c=2+2116&aid=987
Effects of Pesticides. Global Healing Center Natural Health & Organic Living. November 15,
2012, Retrieved from http://www.globalhealingcenter.com/effects-of-pesticides/effects-ofpesticides
Glyphosate-based pesticides affect cell cycle regulation. PudMed.gov. November 30, 2012,
Retrieved from www.ncbi.nlm.nih.gov/pubmed/15182708
Wildlife and Pesticides: A practical guide to reducing the risk, NSDU. November 25, 2012,
Retrieved from http://www.ag.ndsu.edu/pubs/ansci/wildlife/wl1017-1.htm