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8-Methoxypsoralen (8-Mop) and 6,7-dihydroxybergamotin (DHB) added to HA0 yeast can be photocarcinogenic if exposed to prolonged UV ABSTRACT

Grapefruit is known to be unhealthy due to exposure of UV because of the photo carcinogen 6,7-dihydroxybergamotin (DHB) which can lead to melanoma. 8-M`ethoxypsoralen (8-mop) is a drug known to treat skin disease. We can see individual diagnosis of melanoma but we cannot see the effects on a cellular level and we are unsure if 8-mop is a photo carcinogen. If DHB and 8-mop are both photo carcinogens, then we would expect there to be significant cell reduction. We used HA0 yeast with applied vehicle treatment (HA0 and water) of both 8-mop and DHB exposing it to an inhibitory concentration of 50% (IC50). It was evident that both 8-mop and DHB are photo carcinogens due to over 50% of reduction in cell survival compared to the vehicle treatment with no IC50 exposure. This implies that you should not absorb DHB or 8-mop if you spend most of your time outside.

INTRODUCTION

Grapefruit is known to be healthy when absorbed at recommended volumes but can be unhealthy if exposed to ultraviolet radiation (UV) (Wu, 2015). However, the drug responsible for this effect is 6,7-dihydroxybergamotin (DHB) (Kakar, 2007). If you expose grape fruit to UV then the risk of being diagnosed with melanoma increases significantly. Because of this, DHB is likely to be a photo carcinogen however, it is not likely that DHB by itself (no UV exposure) will lead to melanoma (Wu, 2015). Even though we can see a difference on a large scale, it does not show survival at the cellular level. If diagnosed with melanoma, we can use drugs such as of 8-Methoxypsoralen (8-mop) which is known for skin disease treatment (Feng, 2015). However, similar to DHB, 8-mop is a questionable photo carcinogen leading to deleterious activity on the cellular level. Moreover, the effect of drugs are only relative to the dose or concentration absorbed therefore, if we were to add physiological and pharmacological doses of both DHB and 8-mop to a molecular model organism, such as yeast, and expose them to UV, the expectation of cell survival would decrease in both situations. In addition, under no UV exposure, prescription of 8-mop would be a healthy drug treatment for diseases like melanoma.

MATERIALS AND METHODS

We used average UV sensitive HA0 yeast strain purchased from Carolina Supply to determine if there were significant survival effects between 8-mop doses without UV exposure

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and 8-mop doses with UV exposure. We simulated sunlight for inhibitory concentration of 50% (IC50) which is the amount of UV allowed to kill 50% of a colony (Kalliokoski, 2013). We repeated this experiment with DHB then covered the yeast extract-peptone-dextrose medium and adenine (YPAD) plate containing 8-mop with aluminum foil and exposed it to IC50. Three 10 fold dilutions from a “just turbid” treatment, one million cells in water, were done to get two colonies of 1000, 100, and 10 cells at 10µL spots; this was known as the vehicle treatment. Sterile techniques were used throughout and t-tests were used to determine significant survival effects between the drugs by itself, and the drugs plus IC50 exposure.

Setting up the Vehicle, control experiment

Our control consisted of the vehicle treatment (HA0 and water). In prevention of cell division, we arrested the cell cycle by leaving yeast on ice until extraction (Yamagishi, 2010). After our dilution process, we spotted YPAD plates with 8-mop and DHB then incubated at 30°C for two days.

Spotting, 8-Mop and DHB treatment

We compared a pharmacological dose of 50µM 8-Mop to a physiological dose of 500nM 8-mop in order to determine the survival effects between 8-mop by itself and 8-mop plus IC50. We then repeated the same experiment with aluminum foil covering the YPAD plate. Since two colonies were added at each dilution, we took the average cell counts across all dilutions and determined its relative survival (# of cells x 100/ average). We then averaged all relative survivals to get an average survival of cells and survival reduction.

Similarly, we compared a recommended dose (1 pill per 6µL extracellular fluid) to an overdose (20 pills per 6µL extracellular fluid) of DHB in order to determine survival effects between DHB by itself and DHB plus IC50. However, we did not use covering. Cell counts were done to differentiate colony sizes between the vehicle with added DHB and DHB exposed to IC50 (Figure 2).

RESULTS

The vehicle by itself has a relative survival of 100% but a significant difference was seen between the vehicle and vehicle exposed to IC50 (p<0.05). When the pharmacological and physiological dose of 8-mop was added to the vehicle, there was barely any difference in survival reduction (p>0.05). However, incubation of 8-mop amongst both concentrations showed a significant difference when exposed to IC50 (p<0.05). No difference was shown when

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aluminum foil was added to the 8-mop YPAD plates (p>0.05) (Figure 1).

Figure 1. Average cell survival of HA0 amongst all treatments

After incubation of DHB treatment, colonies for recommended dose remained about the same with little reduction of survival (p>0.05) and colonies for overdose was significantly different (p<0.05). However, when IC50 was applied to each dose, over half the colonies were killed (p<0.05) (Figure 3).

Figure 2. More than 50% of cells die out when DHB is exposed to IC50.

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Figure 3. DHB exposure to IC50 kills over 50% yeast. DHB shows no significant difference between vehicle and DHB without UV (P>0.05).

DISCUSSION

It is evident that both DHB and 8-mop are photo carcinogens. In both experiments, over half the colony died due to IC50. This suggests that if you were to drink grapefruit and then go to the beach, you can potentially be diagnosed with melanoma. In addition, if you were prescribed 8-mop to treat melanoma, you are at risk of damaging almost half your cells with UV exposure. Because there was no difference when aluminum foil was applied to 8-mop during IC50 exposure compared to 8-mop without UV, we can suggest that 8-mop by itself does not cause any damage. This suggests that 8-mop is only deleterious with prolonged UV exposure with evidence showing about a 50% reduction of the 500nM dose and over 50% reduction of 50 µM dose when IC50 was applied. In addition, more than 50% of cells died when DHB was exposed to IC50 at both the recommended dose and overdose (Figure 3). Conclusively, no matter what dose you absorb of DHB or 8-mop, you are at high risk of melanoma when exposed to UV.

It would make sense that DHB and 8-mop are not suitable drugs for people who are active outside or constantly exposed to sunlight. Moreover, an overdose of DHB results in cell survival reduction of about 10% without UV exposure (Figure 3). Even though it is a small percentage, overdose of DHB can increase risk of DNA damage resulting in disease without the photo carcinogen effects. It is also evident that comparisons in the colony size can be made between the drugs before and after exposure which is something we could not see with the grapefruit experiment (Figure 2). These comparisons can allow us to test other drugs for similar effects. Since aluminum foil was applied to shield UV, we can test hypothetical UV resistant material such as clothing or metal which could imply suitable situations for 8-mop and DHB prescription. Because data is only evident for prolonged UV exposure, we can also test for

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average UV exposure. This would imply any risk of melanoma for someone who only spends average amount of time outside versus above average amount of time outside per week.

WORKS CITED Feng, M. (2015). Effects of 8-methoxypsoralen on the metabolism of 4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanone in mice. PHARMACOLOGY & PHARMACY.

(This study showed that the enzyme CYP2A5 in mice was determined by the coumarin 7-hydroxylation reaction. This suggested that 8-MOP inhibited CYP2A5 in female C57BL/6 mice.)

Kakar, S. (2007). 6'7'-Dihydroxybergamottin contributes to the grapefruit juice effect. PHARMACOLOGY & PHARMACY.

(This study showed that DHB significantly affects the inhibitory grape juice effect of cytochrome P450 (CYP) 3A4.)

Kalliokoski, T. (2013). Comparability of Mixed IC50 Data - A Statistical Analysis. MULTIDISCIPLINARY SCIENCES.

(This study shows that that mixing IC50 data from different assays adds moderate noise to overall data even if details of assay conditions are not known.)

Wu, S. (2015). Citrus Consumption and Risk of Cutaneous Malignant Melanoma. . Oncology .

(This study showed that citrus consumption increased melanoma risk in two cohorts of men and women.)

Yamagishi, H. (2010). Role of bottom-fermenting brewer's yeast KEX2 in high temperature resistance and poor proliferation at low temperatures. BIOTECHNOLOGY & APPLIED MICROBIOLOGY.

(This study showed that the gene KEX2 is responsible for yeast proliferation at low temperatures which is needed for bottom-fermenting brewer's yeast.)

AKNOWLEDGEMENTS

I would like to thank my lab professor Dr. Stephan Woditschka for purchasing DHB, HA0 yeast, and 8-mop. I would also like to thank Dr. Woditschka for designing and directing the experiment which included overseeing our protocols, applying the incubation process, and counting yeast in preparation and publication of our results. I would like to thank my group members Megan Davitt-Loysen, Molly Love, and Whitney Bond for their hard work piecing together a protocol including the YPAD plate setup, dilution process for the vehicle treatment, and DHB/8-mop spotting. I would also like to thank my colleague Evan Maultsby for his time on peer reviewing.