EFFECT OF LEAD (Pb)

EXPOSURE ON SKIN

ELASTICITY

CHAD-2010

ECOLE PRIVEE

INTERNATIONALE TCHADO-TURQUE

EDITOR and ADVISORY FAYSAL YILMAZ

NTIC Head of Biology Department nigeria_biology@yahoo.com

ASSOCIATE EDITORS

Dayanat Mammadov

EPITT Computer Teacher

Yousouf Daoussa Deby Itno EPITT Student

(4emeClass) Younous Oumar Souni

EPITT Student (4emeClass)

Haggar Bechir Ali Abdramane Haggar EPITT Student

(5emeClass) Idriss Ramadan Erdebou

EPITT Student (4emeClass)

The Effect of Lead (Pb) Exposure on Skin Elasticity

ABSTRACT The goal of this project is to determine whether exposing chicken skin to lead metal for an extended period of time (1 week) in DI water will change the Young’s modulus of the skin. The aim of using lead in this experiment is to be able to apply any findings to real-world situations in which people become exposed to lead through their water pipes. If it is found that lead does have adverse effects on chicken skin, the findings can be used as a basis for further testing with human skin so that the negative health effects of lead can be further understood. This greater understanding could be used to generate more awareness of the possible harms of lead exposure and to develop treatments against lead poisoning. Based on literature findings on the composition of collagen and elastin fibers in the skin, it is hypothesized that lead exposure will make the skin more fragile and prone to breakage than normal, resulting in a lower Young’s modulus than unexposed skin. By comparing the Young’s moduli of the lead-exposed skin samples to the Young’s moduli of unexposed samples using a one-tailed, unpaired t-test in Excel, p<0.05 did reveal that the Young’s modulus of the exposed samples were significantly less than that of the unexposed samples.

INTRODUCTION

The goal of this project is to determine whether exposure to lead can change the elasticity of chicken skin, as roughly measured by Young’s modulus. It is widely known that the extracellular matrix of skin (and many other tissues) contains numerous collagen and elastin fibers. The fibers are constructed from individual collagen or elastin molecules which are covalently cross-linked. The cross-links form between the side chains of different lysine amino acids in the molecules and lend a higher tensile strength to the fibers and hence to the skin.

The lysine side chains have terminal –NH3+ groups which join by forming a bond

between the N atoms (1). In the process, H atoms are given up. However, if a sufficient number of these bonds are broken, the skin will become fragile and tear-prone (1). In order to break the bonds, H atoms must be reintroduced through a reduction reaction. One possible way of accomplishing this reduction might be to expose the tissue to lead, which can undergo the reaction Pb→Pb+2 + 2e-, thereby providing electrons to initiate the reduction reaction and break the bonds.

This project was developed to protect the public health by minimizing lead levels in drinking water. If water is too corrosive, it can cause lead to leach out of the plumbing materials and fixtures and enter the drinking water. Children are especially susceptible to high levels of lead, which can cause damage to the brain, red blood cells, and kidneys. Exposure to low levels of lead can cause low IQ, hearing impairment, reduced attention span and poor classroom performance. High exposure to copper can cause stomach and intestinal distress, liver and kidney damage, and complications from Wilson’s disease in genetically-predisposed people. High lead levels in adults have been linked to high blood pressure. Pregnant women and their fetuses are especially vulnerable to lead exposure, which can significantly harm the fetus, cause low birth weight and slow down normal mental and physical development.

Using lead could lend the experiment a practical objective, since exposing chicken

skin to lead in this experiment could simulate the exposure to lead that some people get from their tap water if the pipes or solders in their homes are old (pre-1987) and lead-containing (City of N`DJAMENA/Chad Water Department). Thus, if it is found that such exposure has the capability of changing the mechanical properties of the skin, further experiments could be done to determine whether human skin is also at risk from such exposure. In this project unexposed skin was used for comparison of the exposed skin.

EQUIPMENTS Major Equipment

Handmade Instron - The Instron was used to stretch the lead-exposed skin samples. The Excel software will record force/displacement data for each sample.

Lab Equipment

2 Large plastic containers- This was required to be large enough to soak chicken legs wrapped in lead sheeting prior to the actual experiment. Ten legs will need to be soaked in each container. Surgical scissors- These were used to separate the skin from the legs and cut out samples from the skin of set dimensions. Knife/scalpel- These were needed to help with cutting the skin off of the leg itself and to cut the skin into sample pieces. Like the marker, they could also be used to make small markings (in the form of cuts) where the skin should then be cut with the scissors. Forceps- These were useful for peeling the skin off the legs and for holding the skin in place when the samples are being cut.

Cutting board- This was used as the surface on which the skin is stripped from the legs and cut into samples

Calipers- These were used to measure the thickness of the skin samples and the distance between the loading clamps on the Instron once samples are loaded.

Weight set (500g, 1kg, 2kg) - These were used to verify that the load cell transducer in the Handmade-Instron is outputting correct measurements by making measurements for known applied loads.

Incubator- It was used to stimulate the human body by adjusting the temperature as 37oC

Supplies

20 freshly slaughtered chicken - The skin was exposed to lead and will act as the actual test subject in this experiment. 20 legs are needed for each group to use 10 in an experiment.

20 1 ft. x 1 ft. (1 ft = 30.48 centimeters) pieces of 1/32 inch (1 inch 2.54 cm) 99% pure lead sheeting- These are needed to act as the source of lead exposure for the skin.

Gloves- These were worn by anyone handling the lead or meat to avoid contamination of their hands by germs from the meat or by lead, which, if ingested, can be harmful.

DI water- This was necessary to soak the lead-covered legs in before the experiment.

Paper towels wet with DI water- These was needed to store the skin samples after they have been cut and before they are loaded into the Instron to prevent their drying out, which could cause changes in their mechanical properties.

Microscopes & Fixed Human Skin Preparets- To be able to understand better the anatomy of human skin, some specimens were observed unde microscopes.

METHODS

1. One week before the lab was to be run, separate the legs from corpse and wrap each of 10 chicken legs tightly in a 1 ft. x 1. ft. piece of lead sheet (maximize the amount of skin that is directly in contact with the lead, especially the region of skin just above the knee). Next, submerge all of the wrapped and non-wrapped legs in two large plastic containers independently and fill with DI water. Set the containers in an incubator (37oC) and let it sit for a week. BE SURE TO WEAR GLOVES DURING THIS STEP AND ALL STEPS IN WHICH THE CHICKEN SKIN OR LEAD IS HANDLED.

1a- Leg separation from corpse

1b- Lead sheet preparation

1c- Wrapping the legs

1d- Submerged two groups of legs

1e- Filling the containers with DI water

1f- Ready legs

1g- Putting the legs in incubator

1h- One week at 37oC

2. On the day of the experiment, set up the handmade-Instron and calibrate it. Generally:

• Verify the load transducer settings using the provided set of known weights. • Make sure the Instron is set to move up, or in other words, to stretch the samples.

2a- Main components of handmade-Instron

2b- Setting up the Instron

2c- Loading known weighs

2d- Stretching the sample

3. Remove the lead from the chicken legs and the skin from the legs. Cut the skin off so that the part above

the outside of the knee remains intact. While not working with the skins, place them in wet paper towels. Make sure you note in what orientation you place them.

3a- Dissection unit

3b- Getting ready for removing the skin

3c- Wetting the paper towel

3d- Cutting the skins off

3e- Placing them on wet paper towels

3f- Skin pieces are ready

4. Cut 2 samples of skin from each of the 20 specimens. The samples should be taken from just above the

outside of the knee and have dimensions of 2 cm x 4 cm (determined to be appropriate for Instron testing). Use the calipers to make measurements. Place the samples back in the paper towels until testing occurs.

4a- Cutting sample skins

4b- Using calipers

4c- Prepared GI (no lead) skins on towel

4d- Prepared GII (with lead) skins on towel

5. Taking one sample, clamp it in the Instron with the 2 cm sides in the clamps. Using the calipers,

measure the distance between the top of the bottom clamp and the bottom of the top clamp. This will act as the gage length in strain calculations and should be kept constant for all samples. Also, measure the width of the sample in the clamps and the thickness near the center of the sample. Multiply these to find the cross-sectional area of the sample. This was used to calculate stress. Pull the dynamometer for different weighs and measure the distance. Record all data.

5a- Clamping the skin

5b- Measuring the thickness & width

6. Pull the dynamometer for different weighs (10-20-30 N) and measure the strain. Record all data and

repeat it for all 40 samples.

6a- Loading sample

6b- Loading weighs

6c- Measuring the strain

6d- Recording data

ANALYSIS 1. Move the raw force (N) and displacement (mm) data for each sample into an Excel worksheet. Divide

all the force data of a sample by the cross-sectional area of that sample and then divide by 1000 to calculate stress data in kPa. Divide the displacement data of the sample by the gage length to obtain stress data. Repeat for all samples. (Stress=Force/Area)

2. Find the Young’s modulus (E) for each of the skin samples. This was the slope of the fit line for the sample. (E= Stress/Strain)

3. Compare the Young’s moduli of the lead-exposed skin samples to the Young’s moduli of unexposed samples using a one-tailed, unpaired t-test in Excel. Since the variance of the Young’s modulus of the unexposed samples is much more than 5% of their mean, unequal variance is assumed. A p<0.05 will reveal that the Young’s modulus of the exposed samples is significantly less than that of the unexposed samples.

YOUNG MODULUS = STRESS/STRAIN

E= (F/A) / (∆L/L)

RESULTS Experimental Results

         STRESS                (F/A) 

STRAIN                                (∆L/L) 

YOUNG MODULUS              (E=STRESS/STRAIN) 

 SAMPLE NUMBER 

THICKNESS   (mm) 

WIDTH   (mm) 

CROSS SECTIONAL 

AREA (A)(mm2) 

1ST     10/A

2ND    20N/A

3RD        

30N/A

GAGE LENGTH L0(mm) 

1ST 

EXTENT LENGTH ∆L(mm) 

2nd 

EXTENT LENGTH ∆L(mm) 

3rd 

EXTENT LENGTH ∆L(mm) 

(E1)  (E2)  (E3) MEAN  (E) 

GROUP‐I         

NO LEAD 

1  0.80  20.00  16.00  0.63  1.25  1.88  28.00  0.80  1.70  3.20  21.88  20.59 16.41 19.62 

2  1.20  20.00  24.00  0.42  0.83  1.25  28.00  0.60  1.50  3.00  19.44  15.56 11.67 15.56 

3  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.75  1.56  3.10  20.74  19.94 15.05 18.58 

4  0.80  20.00  16.00  0.63  1.25  1.88  28.00  0.80  1.70  3.20  21.88  20.59 16.41 19.62 

5  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.75  1.56  3.10  20.74  19.94 15.05 18.58 

6  1.10  20.00  22.00  0.45  0.91  1.36  28.00  0.65  1.60  3.40  19.58  15.91 11.23 15.57 

7  1.20  20.00  24.00  0.42  0.83  1.25  28.00  0.60  1.50  3.00  19.44  15.56 11.67 15.56 

8  1.13  20.00  22.57  0.44  0.89  1.33  28.00  0.68  1.58  3.24  18.24  15.70 11.49 15.14 

9  1.16  20.00  23.29  0.43  0.86  1.29  28.00  0.61  1.50  2.90  19.71  16.03 12.44 16.06 

10  1.20  20.00  24.00  0.42  0.83  1.25  28.00  0.60  1.50  3.00  19.44  15.56 11.67 15.56 

11  1.24  20.00  24.71  0.40  0.81  1.21  28.00  0.54  1.23  2.84  20.98  18.42 11.97 17.12 

12  1.27  20.00  25.43  0.39  0.79  1.18  28.00  0.50  1.00  2.20  22.02  22.02 15.02 19.69 

13  1.31  20.00  26.14  0.38  0.77  1.15  28.00  0.20  0.80  1.95  53.55  26.78 16.48 32.27 

14  1.34  20.00  26.86  0.37  0.74  1.12  28.00  0.19  0.75  1.60  54.87  27.80 19.55 34.07 

15  0.80  20.00  16.00  0.63  1.25  1.88  28.00  0.80  1.70  3.20  21.88  20.59 16.41 19.62 

16  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.75  1.56  3.10  20.74  19.94 15.05 18.58 

17  1.00  20.00  20.00  0.50  1.00  1.50  28.00  0.70  1.20  2.90  20.00  23.33 14.48 19.27 

18  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.75  1.56  3.10  20.74  19.94 15.05 18.58 

19  1.10  20.00  22.00  0.45  0.91  1.36  28.00  0.65  1.60  3.40  19.58  15.91 11.23 15.57 

20  1.30  20.00  26.00  0.38  0.77  1.15  28.00  0.21  0.90  1.96  51.28  23.93 16.48 30.57 

           

   

   

         STRESS                 (F/A) 

STRAIN                                (∆L/L) 

YOUNG MODULUS              (E=STRESS/STRAIN) 

GROUP‐II  WITH LEAD 

SAMPLE NUMBER 

THICKNESS   (mm) 

WIDTH   (mm) 

CROSS SECTIONAL 

AREA (A)(mm2) 

1ST     10/A

2ND    20N/A

3RD        

30N/A

GAGE LENGTH L0(mm) 

1ST 

EXTENT LENGTH ∆L(mm) 

2nd 

EXTENT LENGTH ∆L(mm) 

3rd 

EXTENT LENGTH ∆L(mm) 

(E1)  (E2)  (E3) MEAN  (E) 

22  0.80  20.00  16.00  0.63  1.25  1.88  28.00  0.42  0.82  1.60  41.67  42.68 32.81 39.05 

23  1.10  20.00  22.00  0.45  0.91  1.36  28.00  0.25  0.74  1.40  50.91  34.40 27.27 37.53 

24  1.10  20.00  22.00  0.45  0.91  1.36  28.00  0.25  0.74  1.40  50.91  34.40 27.27 37.53 

25  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.35  0.78  1.55  44.44  39.89 30.11 38.15 

26  0.80  20.00  16.00  0.63  1.25  1.88  28.00  0.42  0.82  1.60  41.67  42.68 32.81 39.05 

27  1.30  20.00  26.00  0.38  0.77  1.15  28.00  0.12  0.46  0.98  89.74  46.82 32.97 56.51 

28  1.40  20.00  28.00  0.36  0.71  1.07  28.00  0.08  0.42  0.85  125.00 47.62 35.29 69.30 

29  1.10  20.00  22.00  0.45  0.91  1.36  28.00  0.25  0.74  1.40  50.91  34.40 27.27 37.53 

30  1.20  20.00  24.00  0.42  0.83  1.25  28.00  0.26  0.90  1.90  44.87  25.93 18.42 29.74 

31  0.80  20.00  16.00  0.63  1.25  1.88  28.00  0.42  0.82  1.60  41.67  42.68 32.81 39.05 

32  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.35  0.78  1.55  44.44  39.89 30.11 38.15 

33  1.00  20.00  20.00  0.50  1.00  1.50  28.00  0.30  0.75  1.20  46.67  37.33 35.00 39.67 

34  1.20  20.00  24.00  0.42  0.83  1.25  28.00  0.26  0.90  1.90  44.87  25.93 18.42 29.74 

35  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.35  0.78  1.55  44.44  39.89 30.11 38.15 

36  0.90  20.00  18.00  0.56  1.11  1.67  28.00  0.35  0.78  1.55  44.44  39.89 30.11 38.15 

37  1.30  20.00  26.00  0.38  0.77  1.15  28.00  0.12  0.46  0.98  89.74  46.82 32.97 56.51 

38  1.20  20.00  24.00  0.42  0.83  1.25  28.00  0.26  0.90  1.90  44.87  25.93 18.42 29.74 

39  1.00  20.00  20.00  0.50  1.00  1.50  28.00  0.30  0.75  1.20  46.67  37.33 35.00 39.67 

40  0.80  20.00  16.00  0.63  1.25  1.88  28.00  0.42  0.82  1.60  41.67  42.68 32.81 39.05 

Table: Results of experiment and data analyzing

Statistical t-Test Results

The results of unpaired t-test

t= 7.91 Standard deviation= 8.08 Degrees of freedom = 38

Group A: Number of items= 20 29.7 29.7 29.7 29.7 37.5 37.5 37.5 38.1 38.1 38.1 38.1 38.1 39.0 39.0 39.0 39.0 39.7 56.5 56.5 69.3 Mean = 40.0 95% confidence interval for Mean: 36.33 thru 43.65 Standard Deviation = 9.92 Hi = 69.3 Low = 29.7 Median = 38.1 Average Absolute Deviation from Median = 5.43

Group B: Number of items= 20 15.1 15.6 15.6 15.6 15.6 15.6 16.1 17.1 18.6 18.6 18.6 18.6 19.3 19.6 19.6 19.6 19.7 30.6 32.3 34.1 Mean = 19.8 95% confidence interval for Mean: 16.12 thru 23.43 Standard Deviation = 5.69 Hi = 34.1 Low = 15.1 Median = 18.6 Average Absolute Deviation from Median = 3.42

Degrees of

Freedom

Probability, p

0.1 0.05 0.01 0.001

28 1.70 2.05 2.76 3.67

29 1.70 2.05 2.76 3.66

30 1.70 2.04 2.75 3.65

40 1.68 2.02 2.70 3.55 60 1.67 2.00 2.66 3.46

120 1.66 1.98 2.62 3.37

infinity 1.65 1.96 2.58 3.29

CONCLUTION Our t value (7.91) was bigger than the table value (2.03). That proves lead exposed chicken skins had

lost their flexibility by showing low Young module values. It was known the effects of lead on internal organs, but any scientific research about skin was not done before. We hope this project would help to understand the effects of lead exposure better and to aware people who still use lead pipe in their installation.

References

(1) Alberts, Bruce, et al. (2002). Molecular Biology of the Cell. (4th ed., pp. 1099,1102). New York: Garland Science.

(2) Zumdahl, Steven S. (2005). Chemical Principles. (5th ed., pp. 468). (3) Boston: Houghton Mifflin Company. (4) Bains, A., Baumann, B., Connie, C., & Wang, E. (2007). Tensile Testing: Elastic Properties. (5) Meeting the Lead Standards. City of Philadelphia Water Department. Retrieved April 23,

2007 from http://www.phila.gov/water/water_quality2.html#bk_leadstandard.