TheDevelopmentalEffectsofNicotineonChickEmbryos

More than 22 million American women smoke and approximately 25% of these women continue to smoke during pregnancy (Lambers and Clark 1996). Cigarette smoke has long been associated with increased health risks to the unborn baby. It contains thousands of different chemicals that could be harmful to various processes of development. One of the most hazardous chemicals in cigarettes is nicotine. Nicotine, the chief alkaloid in tobacco, is known to have adverse effects on body development. Nicotine exposure is known to cause premature birth, growth restriction, premature rupture of membranes, preterm labor, spontaneous abortion, and an increase in heart rate (Lambers and Clark 1996). Nicotine is also known to impair the absorption of calcium, vitamin C and other vitamins and minerals required by a developing fetus (Nash and Persaud 1989). Despite these known risks many pregnant women continue to smoke, exposing the unborn baby to nicotine.

When smokers breath in smoke they draw nicotine into their lungs. The nicotine is then able to enter the bloodstream and other parts of the body. If a woman is pregnant, the nicotine is able to reach the fetus by crossing the placenta and circulating into the baby's blood. Due to the reduction of oxygen and nutrients supplying the fetal tissues, the effects of nicotine can be seen in every trimester from spontaneous abortions in the first trimester to premature delivery, malformations and decreased birth weight in the third (Blair et al. 1996). Although the effects of nicotine are not the same for every organism, most exhibit some retardation of embryonic growth when exposed.

Previous research conducted on rats have provided results illustrating the effects of nicotine on bone growth. Dr. Paulson and a team of scientists from Ohio State University observed reductions in ossification in the femur, forelimb, nasal bone, ribs, and the skull and face (Paulson et al. 1994). Most likely the retardation of bone development is a result of nicotine inhibiting the absorption of calcium in the embryo. Similar research on humans has also been conducted to examine the effects of nicotine on birth weight and body length. Dr. Bardy and his colleagues from the National Public Health Institute inFinland recorded the birth weight and crown-heel length in newborns that had been exposed to nicotine. They found that the exposed newborns were on average 188 g lighter and 10 mm shorter than the nonexposed newborns (Bardy 1993). This result suggests that nicotine causes a retardation in embryonic development.

Additional research has also been done concerning the effects of nicotine on heart rate. Dr. Dienes and a team of scientists examined the effects of nicotine on the cardiovascular system in pregnant women. What they found was that nicotine was associated with anincrease in maternal and fetal heart rate (Dienes 1999). Therefore, nicotine is known not only to affect growth but also the cardiovascular system.

Although the effects of nicotine have been extensively studied in organisms such as humans and rats, there has been little research done using chick embryos. This is difficult to understand considering that the chick is one of the model organisms used in most experiments involving development. Most of the research conducted thus far concerning nicotine and chicks has been by Dr. Gilani from Germany. In one experiment he investigated the effects of nicotine on 48

hour chick embryos. He exposed 48 hour embryos to 1.0 mg of nicotine for several days. The results conclude that there was a high incidence of embryonic death and developmental defects (Gilani 1986).

In our research, we wanted to expand on Gilani's chick experiments and discover the effects of nicotine on the chick embryo. The objective of our research was to try to answer three specific questions. First, does nicotine inhibit the absorption of calcium in chicks and result in suppression of bone growth? Second, does nicotine cause a retardation in development? Lastly, does the addition of nicotine result in an increase in heart rate?

Experimental Design

1st Experiment
30 eggs were collected and incubated for 3 days. 24 of the eggs were used as the nicotine treatment group and the other 6 were used as controls. The nicotine solution applied consisted of 1.0 mg of nicotine and 0.1 mL of Howard's ringers solution. To administer the nicotine solution, a small piece of the shell was removed and the nicotine was added directly on top of the embryo. The egg was taped up and placed back into the 37^oincubator. The controls were treated the same as the treatment group, except they received 0.11 mL of Howard's ringers solution instead of nicotine. Eggs were treated once and monitored for 3 days.

2nd Experiment
30 eggs were collected and incubated for 3 days. 12 of the eggs were administered 0.5 mg of nicotine in 0.1 mL of Howard's ringers solution. The other 12 eggs were administered with 0.25 mg of nicotine and 0.1 mL of Howard's ringers solution. The 6 controls were treated the same as the nicotine treatment group, except they were administered 0.11 mL of Howard's ringers solution. The nicotine solution was injected directly into the yolk of the egg using a 1cc syringe. Eggs were taped up and placed in a 37^o incubator. Eggs were treated every 3 days. Following the first treatment, eggs were monitored everyday for viability. Pictures were taken using an Olympus DP10 microscope and digital camera.

Heart Rate
7 eggs were collected and incubated for 6 days. Using forceps the embryos were removed from the eggs and placed in warm Howard's ringers solution. With fine forceps, the trunk was severed above and below the heart region. The dorsal region of the embryo was removed and the heart left intact. The heart was transferred to fresh warm Howard's ringers solution. 1.0 mg of nicotine was added directly to 4 of the hearts. Heart rate was measured by counting the beats every 15 seconds and then allowing 15 seconds to pass before the next reading. The procedure for the 3 controls was the same, except they were treated with 1.0 mg of saline solution instead of nicotine.

Staining for Limb Cartilage
2, 13 day old nicotine treated embryos and 3, 13 day old control embryos were isolated for staining. Legs and wings were fixed in 5% TCA for 1 hour. The limbs were then washed 3 times with PBS and immersed in Alcian green stain for approximately 18 hours. They were washed 3 times with 70% ethanol, and once with 85% ethanol, 95% ethanol, and 100% ethanol. Limbs were then washed 3 times with methyl salicylate and left in the solution for 4 days. Staining was observed and images were captured using an Olympus DP10 camera.

Results

First Experiment
After treatment with 1.0 mg of nicotine, all 24 chick embryos were dead. The embryos were removed, but no development could be determined. The 6 control embryos were still alive.

Second Experiment
After approximately 24 hours following treatment, 2 of the embryos from the 0.5 mg treatment group were dead. By 48 hours 6 more embryos from the0.5 mg group were dead and 9 embryos from the 0.25 mg group were dead. At 72 hours after treatment, two more embryos, one from each group, had died. When examined, the two embryos revealed developmental retardation in the cranial region, limbs, and eyes. 3 days following the second treatment 3 more embryos from the 0.5 mg group were dead. At this point, all embryos from the 0.5 mg group were dead and 2, 0.25 mg nicotine treated embryos remained. On day 6 following the second treatment both 0.25 mg treated embryos had died. All controls were still viable. Treatment continued until day 13 when the 2 remaining embryos were removed and examined .To prevent further contamination, the experiment was stopped 5 days short because of the presence of mold in 2 of the controls. One embryo (egg #8) exhibited low body length and birth weight when compared to the control group . The second embryo (egg #9) displayed an apparent redness of the skin. Veins appeared to protrude from the surface of the body. All controls exhibited normal development .

Heart Rate
Immediately after nicotine was administered to the hearts in Howard's ringers solution, a dramatic increase in heart rate was observed. Heart rate continued to rise for approximately 120 seconds post treatment. After this time, the heart rate decreased and leveled out at 112 beats per minute (bpm). The control heart, which was treated with saline solution, displayed a constant heart rate range between 100 and 104 bpm. Measurements were stopped at 4 minutes and 10 seconds after the initial treatments. As the heart rates leveled off, the four nicotine treated hearts were on average 10-15 beats higher than the controls

Alcian Green Staining
Alcian green staining revealed the presence of more cartilage in the wings of egg # 8 compared to the controls Legs were too small and underdeveloped to show any conclusive evidence of bone developmentEgg # 9 exhibited comparable bone formation to the controls 

Discussion

From our experiments we can conclude that doses of nicotine between 0.25 mg and 1.0 mg have adverse effects on chick embryo development. All chick embryos treated with 1.0 mg of nicotine were dead within 3 days. This outcome is in accordance with Gilani's observations that exposure to 1.0 mg of nicotine results in high instances of embryonic death.

When the treatment concentration of nicotine was decreased to 0.5 mg and 0.25 mg we still observed high instances of death. It is known that high amounts of nicotine reduces essential nutrients and oxygen supply to fetal tissues (Lambers and Clark 1996). This suggests that any dose of nicotine over 0.25 mg fatally interferes with embryonic development. Although, the treatment population was severely reduced due to casualties, two eggs survived until day 13 of development. Egg #8 exhibited significant underdevelopment compared to the controls. This can be attributed to the fact that nicotine inhibits the absorption of calcium, vitamin C, and essential minerals (Nash and Persaud 1989). Staining with Alcian green confirms this as large amounts of cartilage was observed in egg #8, whereas the control limbs exhibited evidence of further bone development.

Nicotine also has an affect on the circulatory system. Nicotine is a known stimulant that can lead to an increase in heart rate as our evidence suggests. It is also known to narrow the diameter of the blood vessels, thus slowing down the blood flow. Nicotine also increases the tendency of the blood to clot (Lambers and Clark 1996). The red skin color of egg #9 could be a result of these factors. We

speculate that the red skin color could also be attributed to a phenomena known as collateral circulation. Collateral circulation can result from the reduction of blood vessels to the surface of the skin. The reduced supply of nutrients and oxygen to the skin can lead to the branching of new blood vessels, and therefore an increase in the amount of vessels (Thompson et al. 1986). Therefore, the redness of the embryo from egg #9 can be result of not only the slowing of blood and protruding vessels, but also because of an increase in the amount of blood vessels being recruited to the skin surface.

Overall, we have shown that nicotine has adverse effects on chick embryo development. Our experimental evidence for this stems from the fact that 96% of our nicotine treated embryos died. Although, we saw significant malformations in the two surviving embryos, the surviving population size was not large enough to make a conclusive determination of nicotine's effect on developmental malformations.