Zinc Toxicity in Odora Cells

A thesis submitted to the University of Cincinnati Division of Graduate Studies in partial fulfillment of the requirements for the degree of

Master of Science

in the Department of Environmental Health of the College of Medicine by

Heidi Hsieh

A.B. Harvard University August 2011

Committee: Mary Beth Genter, Ph.D. (Chair) Hassane Amlal, Ph.D.

Abstract

Zinc has been touted as a panacea for the common cold. However, there has been some controversy over whether Zicam, an intranasal zinc gluconate gel purported to fight colds, causes anosmia, or the loss of the sense of smell. Historical evidence has shown that zinc sulfate solutions can cause anosmia in humans along with significant damage to the olfactory epithelium in rodents. However, more recent work has claimed to show that zinc gluconate is less toxic than zinc sulfate. Using an in vitro system to compare the toxicity of zinc sulfate and zinc gluconate on immature and mature rat olfactory sensory neurons, it was found that the toxicity of both zinc salts was similar with zinc sulfate being slightly more toxic than zinc gluconate and occurred at significantly lower concentrations than that found in Zicam nasal gel, which strengthens the epidemiological link between intranasal zinc exposure and anosmia. Mechanistic studies disproved the hypothesis that zinc toxicity was caused by inhibition of the HVCN1 proton channel which would have led to acidosis and apoptotic cell death. It was found that these immature rat olfactory sensory neurons are able to maintain their intracellular pH through a

+ + - - Na /H exchanger, specifically NHE1, and a Cl /HCO3 exchanger. Zinc sulfate, at non-toxic levels, had no impact on intracellular pH via proton transport either after acute exposure or after

24 hours incubation with the cells. In conclusion, zinc toxicity is not mediated through an acidification of intracellular pH.

ii iii Acknowledgments

I would like to thank my advisor, Dr. Mary Beth Genter, for all of her support, patience, and understanding throughout the years. Without her guidance and encouragement, I would not have pursued my studies in Toxicology. I would also like to thank my committee member, Dr.

Hassane Amlal, for his guidance and assistance with this project. Additionally, I could not have completed my work without the assistance of Dr. Sarah Pixley, Ms. Tracy Hopkins, Dr. Marina

Gálvez Peralta, Ms. Mansi Krishan, and Ms. Brenda Schumann. I would also like to thank my family and friends for all of their support and encouragement.

iv Table of Contents

Abstract…………………………………………………………………………………...……….ii Acknowledgments………………………………………………………………………………..iv Table of Contents …………………………………………………………………………………v List of Tables & Figures…………………………………………………………………….……vi List of Abbreviations ……………………………………………………………………………vii Introduction and Background …………………………………………………………………….1 Hypothesis ………………………………………………………………………………………...8 Specific Aims……………………………………………………………………………………...8 Experimental Procedures………………………………………………………………………….9 Results……………………………………………………………………………………………13 Discussion………………………………………………………………………………………..17 Conclusion………………………………………………………………………………...……..19 Future Studies……………………………………………………………………………………19 Literature Cited ………………………………………………………………………………….20

v List of Tables and Figures

Table 1. Solutions used for intracellular pH experiments………………………………………..23 Figure 1. Cell viability of undifferentiated Odora cells after 24 hours incubation with salt solutions.…………………………………………………………………………………24 Figure 2. Cell viability of differentiated Odora cells after 24 hours incubation with salt solutions……………………………………………..………………………….………..25 Figure 3. Comparing cell viability of differentiated and undifferentiated Odora cells after 24 hours incubation with zinc salt solutions…………………………………………...……26 Figure 4. Gel showing expression of HVCN1 in Odora cells…………………………………....27 Figure 5. Gel showing expression of NHE1 in Odora cells……………………………………...27 Figure 6. Gel showing lack of expression of NHE2 in Odora cells……………………………...28 Figure 7. Gel showing lack of expression of NHE3 in Odora cells……………………………...28 Figure 8. Gel showing lack of expression of NHE4 in Odora cells……………………………...29 Figure 9. Proton transport in undifferentiated Odora cells………………………………...…….30 Figure 10. Chloride ion transport in undifferentiated Odora cells…………………………….…31 Figure 11. Proton transport in undifferentiated Odora cells treated for 24 hours with 0.1 mM zinc sulfate……………………………………………………………………………………32 Figure 12. Effect of zinc sulfate dose response on pHi recovery after acidification…………....33 Figure 13. Proton transport in differentiated Odora cells treated for 24 hours with 0.1mM zinc sulfate……………………………………………………………………………...……..34 Figure 14. Effect of acute exposure to 0.15mM zinc sulfate on intracellular pH…………….…35 Figure 15. Gel of DNA isolated from differentiated Odora cells incubated for 24 hours with zinc sulfate………………………………………………………………………………….…36

vi List of Abbreviations

ATP adenosine triphosphate

BCECF-AM 2‟,7‟-bis-(2-carboxyethyl-5(6)-carboxyfluorescein) acetoxymethyl ester

CaCl2 calcium chloride cDNA complementary DNA

Cl- chloride ion

CO2 carbon dioxide

DMEM Dulbecco‟s modified Eagle medium

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

FDA U.S. Food and Drug Administration

HBSS Hank‟s balanced salt solution

- HCO3 bicarbonate ion

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HVCN1 hydrogen voltage-gated channel 1

KCl potassium chloride

K2HPO4 potassium hydrogen phosphate

KH2PO4 potassium dihydrogen phosphate

M molar

MgCl2 magnesium chloride

MgSO4 magnesium sulfate mM millimolar

vii mRNA messenger RNA

Na+ sodium ion

NaCl sodium chloride

NADPH nicotinamide adenine dinucleotide phosphate-oxidase

NaHCO3 sodium bicarbonate

NHE1 Na+/H+ exchanger protein 1

NHE2 Na+/H+ exchanger protein 2

NHE3 Na+/H+ exchanger protein 3

NHE4 Na+/H+ exchanger protein 4

NH4Cl ammonium chloride

OTC over-the-counter

PBS phosphate buffered saline

PCR polymerase chain reaction pHi intracellular pH

RNA ribonucleic acid

ROS reactive oxygen species

RPM revolutions per minute

RT-PCR reverse transcription polymerase chain reaction

SLC9 solute carrier family 9

SLC9A1 solute carrier family 9, member 1

SLC9A2 solute carrier family 9, member 2

SLC9A3 solute carrier family 9, member 3

SLC9A4 solute carrier family 9, member 4

viii TMA-Cl trimethylammonium chloride ts temperature sensitive

µL microliter

WGA-HRP wheat germ agglutinin-horseradish peroxidase

ix Introduction and Background

It has long been known that zinc is an essential mineral. On a molecular level, zinc is an essential structural component in many proteins.1 It can behave as an enzymatic cofactor and regulator of DNA transcription.2 Zinc deficiency has been associated with an impaired immune system, learning deficiencies, complications during pregnancy and delivery, and anorexia.3, 4, 5, 6

In zinc-deficient populations such as those found in developing nations, zinc supplementation can be beneficial. It was found in a double-blind placebo-controlled study in West Africa that zinc supplementation helped to reduce morbidity due to diarrhea, while another study in India showed that zinc accelerated the recovery of infants from pneumonia.7,8 When similar studies were repeated among non-zinc deficient populations in developed nations, the results were mixed. For every study similar to the one performed by the Cleveland clinic in 1996, which showed that zinc salt lozenges could shorten recovery time when taken upon the development of symptoms of the common cold, there appears to be another study that contradicts those results, such as the one performed by scientists in Copenhagen in 1990 showing no statistically significant improvement in cold durations for patients using zinc gluconate lozenges.9, 10

However, the most recent meta-analysis of 13 therapeutic trials and 2 preventative trials found that administering zinc supplements, in the form of either syrup, lozenge, or tablets, within 24 hours of onset of cold symptoms helped to shorten the duration and severity of symptoms associated with the common cold.11

There are a plethora of OTC products on the market which use a zinc salt as an active ingredient and claim to help fight colds. Matrixx Initiatives Incorporated, which produces the

Zicam line of products, was one of the first companies to promote zinc-containing products for relief of cold symptoms, with over a dozen products including zinc-containing nasal sprays,

1 lozenges, gel swabs, and oral mists. Most of their products contain either or both zinc acetate, zinc gluconate, in addition to other traditional cold-fighting remedies, such as Vitamin C or

Echinacea. In 1999, Matrixx introduced an intranasal zinc gluconate gel that was supposed to help the user to get over her cold faster. Since Zicam nasal gel was marketed as an OTC remedy

(homeopathic drug), it was not regulated by the U.S. Food and Drug Administration (FDA).

Within a year of releasing the drug on the market, there were reports among the medical community of patients who had used zinc intranasal spray gels and consequently suffered from anosmia, or the loss of the sense of smell. For example, over a three year period, physicians at

University of California at San Diego‟s Nasal Dysfunction Clinic observed seventeen patients who self-reported using zinc gluconate intranasally. Out of the seventeen patients, seven were anosmic, while ten were hyposmic (displaying an impaired sense of smell). These patients reported a burning sensation after applying the gel, and some patients reported olfactory dysfunction more than 6 months after zinc exposure.12 After receiving over 100 complaints from people who became anosmic after using Zicam nasal gel, the FDA issued a consumer alert, warning people that Zicam “may pose a serious risk to consumers who use them.”13 The owners of Zicam were not caught by surprise by the FDA warning, since they had paid over 12 million dollars to settle over 340 lawsuits in 2006 with Zicam users who claimed that the product caused their anosmia. Additionally, they had received over 800 complaints from Zicam users who lost their sense of smell, which they did not share with the FDA.14 By June 2009, Matrixx began a voluntary recall of their Zicam nasal gel, which contained the zinc gluconate salt.

It is not „news‟ in the 1990s and 2000s that zinc salts may have a negative impact on the olfactory system. In 1937, a study was conducted by Dr. Schultz where he intranasally sprayed zinc sulfate solutions in 5000 children in Toronto, Canada as a polio preventative. The

2 prevailing theory at the time was that polio pathogenesis was due to the inhaled virus travelling along olfactory nerves to gain access to the central nervous system and attacking the nerves in the spinal cord. Dr. Schultz believed that by protecting the nose, it would be possible to prevent polio. However, this was not the case and no benefit was seen in polio prevention; rather, many months later, doctors were complaining to Dr. Schultz that approximately 10-13% of their child patients who had received the intranasal zinc spray were suffering from a “complete and permanent loss of smell” as measured by their ability to detect oil of clove and oil of spearmint.15

One of the challenges with accepting these data at face value is the fact that much of the zinc exposure is self-reported with no details on dosage or exposure. When one looks at experiments performed in a laboratory setting, a clearer picture emerges. Dr. Joseph Harding was one of the pioneers in understanding zinc‟s effect on olfactory pathways in mice. Through his work, he found that an intranasal irrigation with 0.17M (equivalent to 5%) zinc sulfate heptahydrate would cause drastic biochemical and morphological changes in the olfactory tissue of mice. He measured the synthesis and transport of carnosine by applying radio-labeled β- alanine to the nares of a mouse. Carnosine is synthesized from β-alanine and histidine and transported from the olfactory epithelium to the olfactory bulb. Dr. Harding found that two weeks after the zinc sulfate irrigation, there was no synthesis and transport of carnosine, while after 16 weeks there was some recovery of synthesis and transport but at a much lower level than in control mice. He found that this was due to the necrosis and sloughing off of the olfactory epithelium and consequently the olfactory receptors that are found within. After a year, the olfactory epithelium does recover, but it is not as thick or as densely populated with olfactory receptors as those in control mice. A more startling change was the reduction in size of the olfactory bulb (~48% of the weight of control) even 16 months after exposure. All of these

3 biochemical and morphological changes resulted in a complete loss in ability to find food pellets within two minutes up to 2 weeks after exposure. There was gradual recovery after 6 weeks which culminated in 63% of mice being able to find their food 6 months after the zinc irrigation.16 As a result, intranasal zinc sulfate irrigation is used as the standard to cause anosmia in mice without having to resort to an invasive surgery like olfactory nerve transaction or olfactory bulbectomy.

As concern rose in the medical community about Zicam nasal gel causing anosmia, a paper was published by Dr. Burton Slotnick where he concluded that “moderate volumes [of zinc gluconate nasal spray], even those far in excess of a recommended dose, were largely without effect on odor detection and discrimination tasks. These outcomes fail to support the claims from recent clinical case reports that use of a zinc gluconate-containing nasal spray can produce anosmia.”17 Dr. Slotnick intranasally injected three different volumes (2 µL, 8 µL, 50 µL) of either Zicam (1.6% zinc gluconate) or 5% zinc sulfate solution with 50 µL of saline as a control using a microliter syringe, which was inserted into the mouse‟s snout past the external naris. Dr.

Slotnick showed that the two lower volumes of zinc gluconate did not impair transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from the olfactory epithelium to the olfactory bulb, while the 50 µL solution showed impairment at 3 days with almost complete recovery after 21 days. WGA-HRP transport relies on intact olfactory neurons, spanning the entire distance from the nasal cavity to the olfactory bulb. In the zinc sulfate-treated mice, the 2

µL-treated mice showed light or moderate level of damage, while after 11 days, the 50 µL- treated mice still had no detectable transport. Dr. Slotnick used a different method of assessing behavioral responses in his mice compared to Dr. Harding‟s work. The mice in Dr. Slotnick‟s paper had been trained to detect ethyl acetate and discriminate between ethyl acetate and propyl

4 acetate. He found that mice treated with the two lower volumes of zinc gluconate solution performed as well as control mice on both ethyl acetate detection and two-odor discrimination, while the 50 µL zinc gluconate-treated mice showed impaired performance but recovered by day

14 or 18 which did not occur in the 50 µL zinc sulfate-treated mice. Dr. Slotnick concluded from these data that the zinc sulfate treatment always has a markedly greater effect than the

Zicam product due to the higher zinc concentration and the possibility that the gluconate salt is less toxic than the sulfate salt. In the discussion section of the paper, Dr. Slotnick also notes the historical literature on the “polio vaccinations” is not relevant to the Zicam case due to the use of devices to make contact with the olfactory epithelium farther back in the nose which the Zicam device would not enable. Also, he notes that the method of olfactory assessment in the “polio vaccination” reports was much more crude compared to contemporary standards and did not have any long-term follow up on the patients.18

With regard to Dr. Slotnick‟s paper, there are some points that need to be raised. First, there is the question of bias, since the work was funded by a grant from Matrixx. Second, the lack of toxicity or impaired function of the 2 µL zinc sulfate-treated mice raises the question on whether any solution of such low volume actually reaches the olfactory epithelium. In his paper,

Dr. Slotnick shows that a 2 µL injection of the WGA-HRP reaches the olfactory bulb but this does not prove that there was significant exposure of the olfactory epithelium to the test solutions. In his methods, there is an explanation on how the volumes tested were calculated based on the volume of the human nose such that relevant exposures would be used. While the nasal volume and volume of Zicam product used may be relevant, the structure and tissue distribution of rodent noses is different enough from humans that it may be difficult to extrapolate dosage based on nasal volume alone. Additionally, as discussed in Dhuria‟s review,

5 a small volume exposure, such as 2 µL, results in deposition mainly in the respiratory epithelium with little of the tested solution reaching the olfactory epithelium, thus raising the question as to whether the exposure in Dr. Slotnick‟s work is significant.19 There is also an anomaly in the data which Dr. Slotnick does not address in that the 8 µL zinc sulfate-treated group actually performed better than the 50 µL Zicam-treated mice on all the detection and discrimination tests, despite his claims that the Zicam treatment is less toxic than the zinc sulfate treatment at all levels.

Regardless of the conflicting evidence on rodents under controlled conditions, the most compelling evidence can often be derived from carefully-controlled epidemiological studies. In

2010, the same physicians at University of California at San Diego who had published a paper on their 15 anosmic patients (Davidson and Smith), combined the available published clinical, biological and experimental data and analyzed it using the Bradford-Hill criteria to establish whether there was sufficient data to demonstrate a causal link between intranasal zinc gluconate usage and anosmia or hyposmia.20 Much of their human evidence was based on their own patients, case reports from different physicians located in different cities in the United States, and historical evidence such as the work done by Dr. Schultz in Canada. The authors noted that there are limitations to their analysis, such as the lack of experimental evidence in humans under controlled conditions showing a causal relationship between zinc and anosmia, which could be remedied by a large-scale randomized trial in accordance with FDA regulations, but due to ethical considerations this would be impossible, and the potential selection bias due to the

„observational nature of the 4 studies linking cationic zinc to anosmia‟. Despite these and other limitations, the authors make a very strong case for a causal relationship between intranasal zinc and anosmia.

6 There are many proposed mechanisms for zinc-induced toxicity. One potential mechanism is the inhibition of glutathione reductase. It was observed in astrocytes that exposure to greater than 100 mM zinc acetate results in the inactivation of glutathione reductase via an

NADPH-dependent mechanism, while 150 mM zinc acetate results in cell death.21 Another mechanism is via the zinc cation slowing the clearance of peroxides and promoting the intracellular production of reactive oxygen species (ROS), which the authors believe is the driving mechanism of cell toxicity.21 The production of ROS, which can also cause oxidative stress, DNA damage, and ATP loss, which can all trigger apoptosis lends credence to the hypothesis that zinc can induce apoptosis, which has been observed via DNA fragmentation and release of cytochrome c from the mitochondria.22, 23 However, other papers showed that free radical scavengers, such as superoxide dismutase and catalase, did not protect against zinc toxicity.24 It has been suggested that zinc-induced apoptosis may be dose-dependent with lower levels of zinc inducing apoptosis while higher concentrations cause necrosis.25, 26 However, other scientists have found that zinc can provide a protective benefit against cadmium-induced apoptosis at lower concentrations, while inducing apoptosis at more elevated levels in C6 rat glioma cells.27 Another potential mechanism may be zinc‟s ability to inhibit the hydrogen voltage-gated channel 1 (HVCN1) which prevents the cell from exporting excess protons.28 This results in the cell becoming acidic which is known to cause apoptosis.29, 30, 31 It appears that the mechanism of zinc toxicity is more complicated than it would appear and is affected by many factors, such as concentration of zinc tested, the length of exposure, the cell type, and the presence of other toxic chemistries.

7 Hypothesis

Based upon the literature, it was hypothesized that zinc salts were toxic to olfactory neurons regardless of the cation present in the salts. Additionally, it was hypothesized that zinc toxicity is due to acidosis of the cells caused by zinc inhibiting the HVCN1 channel which prevents the export of protons.

Specific Aims

1. Determine whether there is a difference in toxicity between zinc gluconate and zinc

sulfate, using the rat olfactory neuronal cell line Odora.

2. Determine whether mechanism of zinc toxicity is due to inhibition of HVCN1 protein

channel and thus intracellular pH.

8 Experimental Procedures

Odora cell line. The Odora cell line, developed by Dr. Dale Hunter to study interactions between odorants and olfactory neurons, was used in the present studies.32 The Odora cell line was derived from rat olfactory epithelium transfected with a temperature-sensitive (ts) mutant of the SV40 large T antigen. When the cells are grown at 33°C, they resemble immature olfactory neurons; however, when they are grown at 39°C in the presence of dopamine and ascorbic acid

(below), the tsA58-Tag degrades, allowing the cells to differentiate into mature olfactory neurons. Mature Odoras can be identified by their morphology (thin and flat, as compared to rounded for the immature Odoras); the expression of functional olfactory receptors on the cell surface; and the expression of GAP-43, which is a nervous tissue-specific cytoplasmic protein that is associated with neurite formation and plasticity.33

Cell culture. Odoras were cultured at 33°C with 5% CO2 in normal media (DMEM, containing

4500 ppm glucose, without sodium pyruvate; Hyclone) supplemented with 10% fetal calf serum,

1% glutamine, and 1% pen/strep/fungizone. In order to differentiate the cells, the cells are grown to 30% confluence at 33°C before being transferred to an incubator at 39°C with 7% CO2 and adding the following chemicals, to the normal media described above to achieve the following final concentrations in the differentiation media: 1 μg/mL insulin, 20 μM dopamine, and 100 μM ascorbic acid. Cells were passaged by rinsing the flask three times with Hank‟s

Balanced Salt Solution (HBSS; Hyclone) then treating with trypsin/EDTA for 5 minutes in 37°C incubator. Trypsinization was stopped by adding 5x volume normal media, as described above, and centrifuging the solution at 1300 RPM for 5 minutes. The pellet was resuspended in normal media and counted using Trypan blue and a hemocytometer.

9 Treatment with salt solutions. Sterile 10 mM and 2 mM stock solutions of the four salts tested

(zinc sulfate, zinc gluconate, sodium sulfate, and sodium gluconate) were prepared by dissolving the salts in distilled water and filtering the solution through a 0.2 μm syringe filter, which was then added to the media to create the salt solutions that were used to dose the cells.

Undifferentiated and differentiated Odora cells were treated with the respective salt solutions at the following final concentrations: 0.01 mM, 0.025 mM, 0.05 mM, 0.075 mM, 0.1 mM, 0.2 mM,

0.3 mM, 0.4 mM, and 0.5 mM. As controls for the sulfate and gluconate cations, Odoras were treated with sodium salt solutions at the following concentrations: 0.05 mM, 0.1 mM, 0.2 mM,

0.3 mM, 0.4 mM, and 0.5 mM.

Cell viability assay for undifferentiated cells. Crystal violet was used to estimate cell viability as previously described.34 Cells were seeded in a 96-well plate with 25,000 cells in 100 μL of normal media in each well. The cells were allowed to grow for 24 hours before the addition of the salt solutions. The cells were allowed to grow for an additional 24 hours before the reaction was stopped by removing the media, and adding 100 μL of 4% glutaraldehyde in phosphate buffered saline (PBS) for 10 minutes. The wells were rinsed three times with PBS followed by the addition of 100 μL of 0.1% crystal violet in water for 30 minutes. The wells were rinsed three times with water and allowed to air dry for 24 hours in a hood. Ten percent acetic acid

(100 μL) in water was added to each well and rocked for at least 10 minutes or until all the crystal violet was dissolved. The 96-well plate was read at 540 nm with the lid removed using the EL800 Universal Plate Reader (Bio-Tek Instruments) and the KCJunior program.

10 Cell viability assay for differentiated cells. Cells were seeded in a 96-well plate with 10,000 cells in 100 μL of differentiation media in each well. The cells were allowed to grow for 48 hours before the addition of 100 μL of salt solution, which is created by combining either 2 mM or 10 mM salt solution with differentiation media. The cells were allowed to grow for an additional 24 hours before the reaction was stopped by removing the media. The cell fixing, staining, and plate reading is the same as for the assay for undifferentiated cells.

RNA isolation, cDNA synthesis, and PCR of Odora cells. Undifferentiated Odoras were cultured in T75 flasks to 90% confluence and rinsed three times with HBSS before being harvested by scraping from the bottom of the flask. The cells were suspended in normal media and centrifuged at 1000 RPM for 5 minutes. The pellet was homogenized in 1 mL of TRI Reagent

(Molecular Research Center), and RNA was isolated per the manufacturer‟s protocol. RNA concentration was measured by nanodrop ND-1000 Full-spectrum UV/Vis spectrophotometer.

Total RNA (1 ng) was reserve transcribed using Verso cDNA kit (Thermo Scientific). The resulting cDNA products were amplified using Taq PCR Master Mix Kit (Qiagen) and the following primers, mouse HVCN1, NHE1, NHE2, NHE3, and NHE4 (see Borensztein P et al. for primer sequence).35 Differentiated Odoras were cultured in T75 flasks to 90% confluence and rinsed three times with HBSS before being harvested by the addition of 2 mL TRI Reagent to the flask. The RNA and cDNA were isolated as described above. PCR products were separated on

1% agarose gels and visualized with ethidium bromide using UV light box.

Intracellular pH monitoring in undifferentiated cells. Intracellular pH was monitored as described by Amlal et al. Briefly, cells at a concentration of 800,000 in 4 mL of normal media

11 were grown on 3.1 x 1.3 cm glass coverslips in 60 x 15 mm polystyrene petri dishes for 64 hours.

Two coverslips were left undisturbed as a control. The other coverslips were treated with either

40 μL of distilled water or 10 mM zinc sulfate solution to create a final concentration of

0.05mM, 0.1mM, 0.15mM zinc sulfate in the dish and then allowed to grow for an additional 24 hours. The cells on the coverslips were rinsed twice with a solution containing 140 mM NaCl, 3 mM KCl, 0.8 mM K2HPO4, 0.2 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, 1 mM MgSO4, 10 mM HEPES, and 5mM glucose. Cells were then incubated with 5 μM BCECF-AM in DMSO in the aforementioned solution for 10 minutes and rinsed with aforementioned solution to remove extracellular BCECF-AM. The coverslip containing the cells was positioned diagonally in a cuvette and placed in a thermostatically controlled holding chamber (37°C) in a Delta Scan dual excitation spectrofluoremeter (PTI, Brunswick, NJ) and perfused with the appropriate solution

(Table 1). The fluorescence ratio at excitation wavelengths of 500 and 450 nm (F500/F450) was used to monitor intracellular pH. The emission wavelength was recorded at 525 nm.36

12 Results

Cell viability in undifferentiated and differentiated cells. The A540 by crystal violet directly correlates with the number of viable cells.34 By normalizing the absorbance of the various treatments to the control, for undifferentiated Odora cells, significant toxicity (i.e. 80% of control or less) occurs between 0.1 and 0.2 mM zinc sulfate and between 0.2 and 0.3 mM zinc gluconate.

The sodium salts appear to have no negative impact on cell viability when tested up to concentrations of 0.5 mM (Figure 1). For differentiated Odora cells, there is significant toxicity between 0.1 and 0.2 mM zinc sulfate and between 0.3 and 0.4 mM zinc gluconate (Figure 2).

Sodium gluconate and sulfate were similarly non-toxic to the differentiated Odora cells over this concentration range (not shown); these solutions appear to have no negative impact on cell viability (Figure 2). When the zinc treatments for both the differentiated and undifferentiated

Odora cells are plotted on the same graph, the zinc sulfate treatments behave similarly, while the zinc gluconate treatment appears to be slightly more toxic to the undifferentiated cells (Figure 3).

PCR evaluation of expression of HVCN1 proton channel. HVCN1 proton channel is inhibited by extracellular zinc, which limits the cell‟s ability to export protons. This proton channel does not require a counter ion for transport and has been found in human nasal epithelial cells, where it helps the body maintain the extracellular pH in nasal mucosa.37 RT-PCR analysis revealed that mRNA encoding for the HVCN1 channel is present in Odora cells (Figure 4).

Expression of Na+/H+ exchanger (NHE) isoforms. The SLC9 family are Na+/H+ exchanger proteins that are found throughout the body. They are responsible for intracellular pH regulation and consequently, cell proliferation and death, and thus, are well conserved among various

13 mammalian species, such as humans, mice, and rats. The SLC9 family has several isoforms encoded by different genes and the mostly abundant isoforms are NHE1 (encoded by SLC9A1), which is the most commonly expressed and is found in all tissues, while NHE2 (encoded by

SLC9A2), NHE3 (encoded by SLC9A3), and NHE4 (encoded by SLC9A4) are generally found in kidney or gastrointestinal tract.38, 39 PCR data depicted in figure 5 indicates that only mRNA encoding for the NHE1 protein is present in Odora cells (Figure 5), whereas NHE2, NHE3 and

NHE4 are not expressed in Odora cells (Figures 6, 7, 8). As expected, all NHE isoforms mRNA are detected in the kidney, which was used as a positive control (figures 5, 6, 7 and 8).

Proton transport in undifferentiated cells. To determine whether undifferentiated Odora cells could export protons independently of Na+ ion, the cells were grown to 80% confluence on coverslips and monitored for intracellular pH (pHi) recovery after an acid load induced using

+ NH4 /NH3 pre-pulse as described by Amlal et al. in the absence or presence of Na+ ion in the

36 perfusion solution (Table 1). Upon equilibration of pHi in solution A, the cells were perfused with solution B containing 20 mM ammonium chloride which caused the pHi to become alkaline.

After the cells reach equilibrium, the cells were perfused with solution C, which was devoid of

Na+. The intracellular pH dropped dramatically and remained acidic until cells were exposed to a Na+-containing solution, which initiated a sharp recovery of cell pH to around baseline level as indicated in figure 9. This result clearly indicate that Odora cells are devoid of Na+-independent

H+ transport mechanisms even after severe cell acidification, and that NHE1 is likely the only

H+ transport mechanism expressed in these cells.

14 - - Presence of a Cl /HCO3 exchanger in undifferentiated cells. To determine whether in addition to

- - NHE1, undifferentiated Odora cells express a Cl /HCO3 exchanger which also could contribute to the regulation of intracellular pH of Odora cells, we used the classical protocol which is based on exposing cells to various solutions which either do or do not contain chloride ions in the

- presence or absence of HCO3 in the solution. Accordingly, cells were grown to 80% confluence on coverslips and loaded with BCECF-AM before being monitored for pHi changes in various

- Cl solutions (solution D, E, F, G; Table 1). Upon equilibration of pHi in solution D, exposure of cells to a nil Cl- solution (Solution E) caused a rise in intracellular pH, indicating that Cl- efflux is

- - accompanied by HCO3 influx. After the pHi reached a plateau, the cells were perfused with Cl - containing solution (Solution D) and the cells acidified with pHi returning to the baseline level,

- indicating that the influx of Cl- is exchanged for the efflux of HCO3 . When the same experiment was run in the absence of bicarbonate (solution F and G, Table 1), no changes in pHi

- - were observed (Figure 10). These results indicate that Odora cells express an active Cl /HCO3 exchanger, which has yet to be identified.

Effect of 24 hour exposure to 0.05 mM, 0.1 mM, and 0.15 mM zinc sulfate on proton transport in undifferentiated cells. Cells were grown to 70% confluence and incubated for 24 hours with 0.05 mM, 0.1 mM, or 0.15 mM zinc sulfate (non-toxic levels as determined by cell viability assay), and then monitored for pHi changes using the same series of solutions as described in proton transport in undifferentiated cells. The slope of initial pHi recovery was calculated for each of the zinc sulfate exposures and normalized to the control. It was observed that there is no significant difference in rate of initial pHi recovery after acidification for any of the zinc sulfate-treated cells

(Figure 11 and 12).

15

Effect of 24 hour exposure to 0.1 mM zinc sulfate on proton transport in differentiated cells.

Cells were grown to 70% confluence and incubated for 24 hours with 0.1 mM zinc sulfate, (a non-toxic level as determined by cell viability assay), and then monitored for pHi changes using the same series of solutions as described in proton transport in undifferentiated cells. It was observed that there is no difference in initial pHi recovery after cell acidification between zinc- treated cells and control (Figure 13).

Effect of acute exposure to 0.15 mM zinc sulfate on pHi in undifferentiated cells. Cells were

+ grown to 80% confluence and perfused with Na solution (solution A) to measure initial pHi.

After equilibrium was reached, the solution was switched to a solution H, which contained 0.15 mM zinc sulfate. It was observed that no change in pHi occurred (Figure 14).

Does exposure to zinc sulfate cause apoptosis in differentiated and undifferentiated cells? Cells were grown to 100% confluence and incubated for 24 hours with zinc sulfate at the following concentrations: 0.075 mM, 0.1 mM, 0.2 mM, 0.3 mM. The cells were lysed and the DNA isolated (Wizard Genomic DNA Purification Kit, Promega). DNA (2g) was run on an agarose gel. No evidence of DNA laddering was observed in any of the treatments for both types of cells

(Figure 15).

16 Discussion

The differences in cell viability between the zinc sulfate and zinc gluconate salt solutions are noticeable and the anion may be playing a role despite not appearing to impact cell survival when administered to Odora cells in the respective sodium salt solutions. This suggests that there may be some credence to Dr. Slotnick‟s claim that zinc gluconate is less toxic than zinc sulfate; however, recall that zinc toxicity in differentiated Odora cells occurred between 0.3 to

0.4 mM zinc gluconate, while the level of zinc gluconate in the Zicam nasal gel formulation, is

1.6% zinc gluconate which equals 35 mM zinc gluconate according to Dr. Slotnick.17

Additionally, when the patent was filed on the original formulation, the inventors note that “at least a 20mM ionic zinc is preferred in the composition to insure that a sufficiently high concentration of ionic zinc is produced by the composition.”40 The concentration of zinc that induces toxicity in Odora cells, which are immortalized rat olfactory neurons, is significantly lower by orders of magnitude than that used by the Zicam nasal gel or in Dr. Slotnick‟s work suggesting that the product may indeed cause significant damage to olfactory tissue when used.

While PCR results suggested the expression of the HVCN1 proton channel in Odora cells

(Figure 4), this proton channel is either not expressed at the cell surface or is non-functional.

This question remains unanswered at this time because of the lack of a high quality antibody for use in Western blots or immunohistochemistry. The absence of a functional HVCN1 proton channel and Na+-indendent proton exchanger was inferred by the lack of recovery in intracellular pH when the Odora cells were acidified in the absence of Na+ ions. If the HVCN1 proton channel or Na+-indendent proton exchanger was present, then the cells would have alkalinized to their initial intracellular pH – this never occurred. These results suggest that the NHE1 exchanger is present and functioning in both undifferentiated and differentiated Odora cells since

17 + recovery to initial pHi was observed when the cells were perfused with a Na –containing solution after acidification. Odora cells are also able to respond to alkalinization of intracellular

- - pH via a HCO3 /Cl -dependent transporter, whose identity is still yet unknown. Due to the lack of effect on initial pHi recovery from 24 hours incubation with zinc sulfate and acute exposure to

0.15 mM zinc sulfate, the mechanism of zinc toxicity on Odora cells is not through an intracellular pH mechanism.

Based upon literature, it was surprising that no evidence of DNA laddering was observed in cells incubated for 24 hours with zinc sulfate suggesting that the cells are not undergoing apoptosis. However, the lack of DNA laddering does not rule out the possibility of apoptosis and further work, such as examining the morphology of the cells and presence of caspase-3/9, must be done.

18 Conclusion

There is a small difference in toxicity as measured by cell viability between zinc sulfate and zinc gluconate; however, the difference is approximately 0.1 mM and 0.2 mM in undifferentiated and differentiated Odora cells, respectively. Despite the presence of HVCN1 in

Odora cells, HVCN1 does not play a role in maintaining intracellular pH. Odora cells express the NHE1 exchanger, which allows it to recover from acidification, and an as of yet unidentified

- - HCO3 /Cl exchanger, which protects it from alkalinization. However, zinc does not affect NHE1 activity or have any impact on intracellular pH after acute exposure. Thus, zinc toxicity is not mediated through an acidification of intracellular pH.

Future Studies

1) Study chloride ion transport in differentiated Odora cells to see whether there is any

difference in their ability to transport ions compared to undifferentiated Odora cells.

2) Study effect of zinc sulfate treatment on undifferentiated Odora cells with respect to

chloride ion transport.

3) Study effect of zinc sulfate and zinc gluconate treatment on differentiated Odora cells

with respect to chloride ion transport.

4) Determine whether zinc sulfate induces apoptosis in undifferentiated and differentiated

Odora cells.

19 Literature Cited

1. Vallee BL. Zinc: biochemistry, physiology, toxicology and clinical pathology. Biofactors. 1988 Jan; 1(1): 31-6. 2. Valee BL and Falchuk KH. The biochemical basis of zinc physiology. Physiological reviews. 1993; 73(1): 79-118. 3. Hambidge KM and Krebs NF. Zinc deficiency: a special challenge. J Nutr. 2007 Apr; 137(4): 1101-5. 4. Golub MS, Keen CL, Gershwin ME, Hendrickx AG. Developmental Zinc Deficiency and Behavior. J Nutrition. 1995; 125(8): 2263S-2271S. 5. Favier AE. The role of zinc in reproduction. Hormonal mechanisms. Biol Trace Elem Res. 1992; 32: 363-381. 6. Katz RL, Keen CL, Litt IF, Hurley LS, Kellams-Harrison KM, Glader LJ. Zinc deficiency in anorexia nervosa. J Adolescent Health Care. 1987; 8(5): 400-406. 7. Muller O, Becher H, Balutssen van Zweeden A, Ye Y, Diallo DA, Konate AT, Gbangou A, Kouyate B, Garenne M. Effect of zinc supplementation on malaria and other causes of morbidity in west African children: randomised double blind placebo controlled trial. BMJ 2001; 322: 1567-1572. 8. Brooks WA, Yunus M, Satosham M, Wahed MA, Nahar K, Yeasmin S, Black RE. Zinc for severe pneumonia in very young children: double-blind placebo-controlled trial. Lancet. 2004 May 22; 363(9422): 1683-8. 9. Mossad SB, et al. Zinc gluconate lozenges for treating the common cold. A randomized, double-blind placebo-controlled study. Ann Intern Med. 1996; 125(2): 81-88. 10. Weismann K, Jakobsen JP, Weismann JE, Hammer UM, Nyholm SM, Hansen B, Lomholt KE, Schmidt K. Zinc gluconate lozenges for common cold. A double-blind clinical trial. Dan Med Bull. 1990; 37: 279-81. 11. Singh M, Das RR. Zinc for the common cold. Cochrane Database of Systematic Reviews 2011, Issue 2. Art. No.: CD001364. 12. Alexander TH. et al. Intranasal Zinc and Anosmia: The Zinc-Induced Anosmia Syndrome. Laryngoscope. 2006; 116(2): 217-220. 13. “FDA Advises Consumers Not To Use Certain Zicam Cold Remedies.” United States Food and Drug Admin. June 16, 2009. 14. Harris G. “F.D.A. Warns Against Use of Popular Cold Remedy.” The New York Times. 16 June 2009. 15. Jafek BW, Linschoten MR, Murrow BW. Anosmia after Intranasal Zinc Gluconate Use. Am J Rhinol. 2004; 18: 137-141. 16. Harding, JW, et al. Denervation of the primary olfactory pathway in mice. V. Long-term effect of intranasal ZnSO4 irrigation on behavior, biochemistry, and morphology. Brain Research. 1978; 140: 271-285. 17. Slotnick B. et al. Olfaction and olfactory epithelium in mice treated with zinc gluconate. Laryngoscope. 2007 Apr;117(4):743-9. 18. Ibid. 19. Dhuria S, Hanson LR, Frey WH. Intransal Delivery to the Central Nervous System: Mechanisms and Experimental Considerations. J Pharm Sci. 2010; 99(4): 1654-1673. 20. Davidson TM and Smith WM. The Bradford Hill Criteria and Zinc-Induced Anosmia. A Causality Analysis. Arch Otolaryngol Head Neck Surg. 2010 July; 136(7): 673-676.

20 21. Bishop GM. et al. Zinc stimulates the production of toxic reactive oxygen species (ROS) and inhibits glutathione reductase in astrocytes. Free Radical Biology and Medicine. 2007; 42(8): 1222-1230. 22. Rudolf R. Depletion of ATP and oxidative stress underlie zinc-induced cell injury. Acta Medica. 2007; 50(1): 43-49. 23. Feng P, Li TL, Guan ZX, Franklin RB, and Costello LC. Direct Effect of Zinc on Mitochondrial Apoptogenesis in Prostate Cells. The Prostate. 2002; 52: 311-318. 24. Borovansky J and Riley P. Cytotoxicity of zinc in vitro. Chem Biol Interactions. 1989; 69: 279-291. 25. Haase H, Watjen W. and Beyersmann D. Zinc induces apoptosis that can be suppressed by Lanthanum in C6 rat glioma cells. Biol Chem. 2001. 382: 1227-1234. 26. Iitaka M, Kakinuma S,Fujimaki S,OosugaI, Fujita T, Yamanaka K, Wada S, and Katayama S. Induction of apoptosis and necrosis by zinc in human thyroid cancer cell lines. J Endocrinology. 2001. 169: 417-424. 27. Wätjen W, Haase H, Biagioli M, Beyersmann D. Induction of apoptosis in mammalian cells by cadmium and zinc. Environ Health Perspect. 2002. 110 Suppl 5: 865-7. 28. Iovannisci D, Illek B, Fischer H. Function of the HVCN1 proton channel in airway epithelia and a naturally occurring mutation, M91T. J Gen Physiol. 2010 Jul; 136(1): 35- 46. 29. McCarty MF and Whitaker J. Manipulating tumor Acidification as a Cancer Treatment Strategy. Altern Med Rev. 2010 Sep;15(3):264-72. 30. De Milito A, Iessi E, Logozzi M, Lozupone F, Spada M, Marino ML, Federici C, Perdicchio M, Matarrese P, Lugini L, Nilsson A, Fais S. Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species. Cancer Res. 2007 Jun 1;67(11):5408-17. 31. Di Sario A, Bendia E, Omenetti A, De Minicis S, Marzioni M, Kleemann HW, Candelaresi C, Saccomanno S, Alpini G, Benedetti A. Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells. Dig Liver Dis. 2007 Jan; 39(1): 60-9. 32. Murrell JR, Hunter DD. An Olfactory Sensory Neuron Line, Odora, Properly Targets Olfactory Proteins and Responds to Odorants. J Neurosci. 1999 Oct 1; 19(19): 8260-70. 33. Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 1997 February; 20(2): 84–91. 34. Kueng W, Silber E, Eppenberg U. Quantification of Cells Cultured on 96-well Plates. Analytical Biochem. 1989; 182: 16-19. 35. Borensztein P, Froissart M, Laghmani K, Bichara M, Paillard M. RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb. Am J Physiol. 1995 Jun; 268(6 Pt 2): F1224-8. 36. Amlal H, Wang Z, Soleimani M. Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells. Am J Physiol. 1997 Oct; 273(4 Pt 1): C1194-205. 37. Capasso M, DeCoursey TE, Dyer MJS. pH regulation and beyond: unanticpated functions for the voltage-gated proton channel, HVCN1. Trends in Cell Bio. 2011. 21(1): 20-28.

21 38. Orlowski J and Grinstein S. The ABC of Solute Carriers: Diveristy of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Archi European J Physiol. 2004 447: 549-565. 39. Beltrán AR, Ramírez MA, Carraro-Lacroix LR, Hiraki Y, Rebouças NA, Malnic G. NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells. Pflugers Arch. 2008 Feb;455(5):799-810. 40. US Patent 6080783 titled “Method and Composition for Delivering Zinc to the Nasal Membrane” filed by RS Davidson, GS Kehoe, LS Kaye in 1998.

22 Table 1. Solutions Used for Intracellular pH Experiments

Solutions Compound A B C D E F G H NaCl 140 120 115 140 140 NH4Cl 20 TMA-Cl 140 NaHCO3 25 25 KCl 3 3 3 3 3 3 K2HPO4 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 KH2PO4 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 CaCl2 1 1 1 1 1 1 MgCl2 1 1 1 1 1 1 MgSO4 1 1 1 1 1 1 1 1 HEPES 10 10 10 10 10 10 10 10 Glucose 5 5 5 5 5 5 5 5 Sodium gluconate 115 140 Potassium gluconate 3 3 Magnesium gluconate 1 1 Calcium gluconate 1 1 Zinc sulfate 0.15 Concentrations are in mM and final pH of solution adjusted to 7.40

23 Figure 1. Cell viability of undifferentiated Odora cells after 24 hours incubation with salt solutions

160% Zinc Sulfate Sodium Sulfate 140% Zinc Gluconate Sodium Gluconate

120%

100%

80%

60% % of control (cells only) (cells control of % 40%

20%

0% cells only 0.01mM 0.025mM 0.05mM 0.075mM 0.1mM 0.2mM 0.3mM 0.4mM 0.5mM n = 6 error bar = standard error of the mean Salt Concentration (mM)

24 Figure 2. Cell viability of differentiated Odora cells after 24 hours incubation with salt solutions

140% Zinc Sulfate Zinc Gluconate 120%

100%

80%

60%

40% % of control (cells only) (cells control of %

20%

0% cells only 0.01mM 0.025mM 0.05 mM 0.075 mM 0.1 mM 0.2 mM 0.3 mM 0.4 mM 0.5 mM n = 6 error bar = standard error of the mean Salt Concentration (mM)

25 Figure 3. Comparing cell viability of differentiated and undifferentiated Odora cells after 24 hours incubation with zinc salt solutions

140% Zinc Sulfate (undifferentiated) Zinc Gluconate (undifferentiated) Zinc Sulfate (differentiated) 120% Zinc Gluconate (differentiated)

100%

80%

60%

% of control (cells only) (cells control of % 40%

20%

0% cells only 0.01mM 0.025mM 0.05mM 0.075mM 0.1mM 0.2mM 0.3mM 0.4mM 0.5mM n = 6 error bar = standard error of the mean Salt Concentration (mM)

26 Figure 4. Gel showing expression of HVCN1 in Odora cells

1 2 3 4 5 6 7 8 9

Lane 1: ladder Lane 2: Olfactory epithelium Lane 3: Nasal respiratory epithelium Lane 4: Nasal respiratory epithelium Lane 5: Kidney Lane 6: Odora Lane 7: Odora Lane 8: Olfactory epithelium Lane 9: Control (water)

Figure 5. Gel showing expression of NHE1 in Odora cells

1 2 3 4 5 6

Lane 1: ladder Lane 2: Control (water) Lane 3: Odora with Reverse Transcriptase Lane 4: Odora without Reverse Transcriptase Lane 5: Kidney Lane 6: Kidney

27 Figure 6. Gel showing lack of expression of NHE2 in Odora cells

1 2 3 4 5 6 7

Lane 1: ladder Lane 2: Control (water) Lane 3: Kidney old sample Lane 4: Kidney with Reverse Transcriptase Lane 5: Kidney without Reverse Transcriptase Lane 6: Odora with Reverse Transcriptase Lane 7: Odora without Reverse Transcriptase

Figure 7. Gel showing lack of expression of NHE3 in Odora cells

1 2 3 4 5 6 7 8

Lane 1: ladder Lane 2: Control (water) Lane 3: Kidney old sample Lane 4: Odora with Reverse Transcriptase Lane 5: Odora without Reverse Transcriptase Lane 6: blank Lane 7: Kidney with Reverse Transcriptase Lane 8: Kidney without Reverse Transcriptase

28 Figure 8. Gel showing lack of expression of NHE4 in Odora cells

1 2 3 4 5 6 7 8

Lane 1: ladder Lane 2: Control (water) Lane 3: Kidney (old) with Reverse Transcriptase Lane 4: Kidney (old) without Reverse Transcriptase Lane 5: Kidney with Reverse Transcriptase Lane 6: Kidney without Reverse Transcriptase Lane 7: Odora with Reverse Transcriptase Lane 8: Odora without Reverse Transcriptase

29 Figure 9. Proton transport in undifferentiated Odora cells

Na+ (A) Na+ (B) TMA+ (C) Na+ (A) + 99 NH4 (B)

88

77

500/450 66 F F

55

44

0 200 400 600 800 1000 1200 33 0 200 400 Time600 (sec) 800 1000 1200

30 Figure 10. Chloride ion transport in undifferentiated Odora cells.

Cl- (D or F) Nil Cl- (E or G) Cl- (D or F) 8.5 Nil Bicarbonate 25 mM Bicarbonate 8

7.5

7

500/450 F F

6.5

6

5.5 0 100 200 300 400 500 600 700 800 Time (sec)

31 Figure 11. Proton transport in undifferentiated Odora cells treated for 24 hours with 0.1 mM Zinc Sulfate

9.5 water vehicle 0.1 mM Zinc Sulfate 8.5

7.5 Slope = 0.0222 6.5

500/450 Slope = 0.0227 F F 5.5

4.5

3.5 0 200 400 600 800 Time (sec)

32 Figure 12. Effect of zinc sulfate dose response on pHi recovery after acidification

140

120

100 dt

/ / 80

500/450 60 dF

40

20

0 0 0.05 0.1 0.15 Concentration of Zinc Sulfate (mM)

33 Figure 13. Proton transport in differentiated Odora cells treated for 24 hours with 0.1mM zinc sulfate.

9 water vehicle 0.1 mM Zinc Sulfate 8

7 Slope = 0.1563 6

Slope = 0.1540 500/450

F F 5

4

3

2 0 200 400 600 800 1000 Time (sec)

34 Figure 14. Effect of acute exposure to 0.15mM zinc sulfate on intracellular pH.

9 Nil Zn+ (A) Zn+ (H)

8.5

8

7.5

7

500/450 6.5 F F 6

5.5

5

4.5

4 0 50 100 150 200 250 300 350 Time (sec)

35 Figure 15. Gel of DNA isolated from differentiated Odora cells incubated for 24 hours with zinc sulfate

1 2 3 4 5 6

Lane 1: ladder Lane 2: Differentiated Odora (control) Lane 3: Odora treated with 0.075 mM Zinc Sulfate Lane 4: Odora treated with 0.1 mM Zinc Sulfate Lane 5: Odora treated with 0.2 mM Zinc Sulfate Lane 6: Odora treated with 0.3 mM Zinc Sulfate

36