Cyclophosphamide-induced disruption of umami taste functions and taste epithelium

Neuroscience 192 (2011) 732–745

CYCLOPHOSPHAMIDE-INDUCED DISRUPTION OF UMAMI TASTE FUNCTIONS AND TASTE EPITHELIUM N. MUKHERJEE AND E. R. DELAY*

monly reported disturbance (41%) followed by sweet (36%), bitter (24%), and sour (21%) (Bernhardson et al., 2007, 2008, 2009). When taste sensitivity was evaluated during chemotherapy, patients showed decreased sensitivity for sweet and sour tastes, but increased sensitivity for bitter taste (Carson and Gormican, 1977; Hall et al., 1980). These changes are a major concern during cancer treatment because they can lead to lower food intake, malnutrition, and poorer prognosis for the afflicted patients (Epstein et al., 2002; Bernhardson et al., 2007, 2008, 2009; Hutton et al., 2007; Halyard, 2009). The prevailing explanation for these changes in taste following chemotherapy is that a patient develops a conditioned taste aversion (CTA) when the profound nausea that accompanies the administration of the drug is paired with food intake (Bovbjerg et al., 1992; Mattes et al., 1987; Bartoshuk, 1990; Ahles and Saykin, 2002). This leads to a generalized aversion for foods, lower consumption, and a general loss of appetitive motivation. This is based on well-established principles of learning (Bouton, 2007) and is well supported in the CTA literature in which cyclophosphamide (CYP) has been used as an unconditioned stimulus in place of lithium chloride (Ader and Cohen, 1975; Bernstein, 1978, 1985; Bernstein and Webster, 1980; Bernstein et al., 1984; MacQueen and Siegel, 1989; Lambert and Whitehouse, 2002). However, the biological effects of chemotherapy drugs on taste epithelium have not been examined closely. The purpose of this study was to explore the effects of CYP on taste behavior and on taste epithelium of mice. CYP was one of the first chemotherapy drugs developed (Aronovitch et al., 1960; Foye et al., 1960; Host and Nissen-Meyer, 1960; Brock, 1989), and has been one of the most commonly prescribed chemotherapy drugs worldwide (Kovacs et al., 1960; Fernbach et al., 1960, 1962; Bagley et al., 1973; Brock, 1989). It has been used primarily in the treatment of leukemia, lymphoma, ovarian cancer, and some malignant tumors (Bagley et al., 1973; Juma et al., 1979a,b,c, 1980, 1984; Cunningham et al., 1988; Schuler et al., 1991; Moore, 1991; Yule et al., 1999; Ludeman, 1999; Malet-Martino et al., 1999; Petros et al., 2002; Pinto et al., 2009; Gor et al., 2010). Currently, it is often used as part of a cocktail of drugs. CYP is a DNA alkylating agent derived from the oxazophorine group. CYP is essentially a prodrug, that is, it is administered in an inactive form, but once inside the body, it is converted into metabolically active products. In hepatic cells, CYP is acted upon by hepatic p450 cytochrome oxidase to form 4-hydroxy CYP (Cohen and Jao, 1970; Juma et al., 1980; Sanderson and Shield, 1996; Colvin, 1999; de Jonge et al.,

Department of Biology and Vermont Chemical Senses Group, 109 Carrigan Drive, Marsh Life Science, University of Vermont, Burlington, VT 05405, USA

Abstract—Clinical studies have reported taste dysfunctions developing in patients undergoing chemotherapy. This adverse side effect is a major concern for the doctors and patients because disrupted taste can reduce appetite, cause malnutrition, delay recovery, and affect quality of life. Cyclophosphamide (CYP) is a common atenoplastic drug used during chemotherapy and is thought to affect taste through learned tasted aversions. This study asked whether CYP also alters umami taste sensory functions and disrupts taste epithelium of mice. Behavioral tests focused on taste acuity, assessed by the ability of mice to discriminate between the taste qualities of two umami substances, monosodium glutamate (MSG) and inosine 5=-monophosphate (IMP), and taste sensitivity, assessed by detection thresholds of MSG and IMP, after an IP injection (75 mg/kg) of CYP. The behavioral results revealed a two-phase disturbance in taste acuity and loss of sensitivity, the first phase occurring within 2– 4 days after injection and the second occurring 9 –12 days after injection. The number of fungiform papillae (with and without pores) decreased immediately after injection and did not begin to recover until 12 days after injection. Circumvallate taste buds began to show disturbances by 8 days after injection and evidence of recovery beginning 12 days after injection. Von Ebner glands were smaller and secreted less saliva 4 days postinjection but not later. These findings suggest the initial behavioral deficits may be because of cytotoxic effects of the drug on taste sensory tissues, whereas the second phase may be because of a disturbance of the taste cell replacement cycle. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: taste cell renewal, cytotoxicity, animal psychophysics, umami, chemotherapy, taste acuity.

Treatments for cancer often involve chemotherapy alone or combined with radiation therapy. Chemotherapy has numerous side effects, including altered taste function. The most frequent disturbances in taste function are hypogeusia (decreased sensitivity), dysgeusia (distortion of taste), or ageusia (absence of taste) (Comeau et al., 2001; Hong et al., 2009). In a study with 518 patients undergoing chemotherapy, 67% reported experiencing some form of taste alteration. Alteration in salt taste was the most com*Corresponding author: Tel: ⫹1-802-656-0455; fax: ⫹1-802-656-2914. E-mail address: [email protected] (E. R. Delay). Abbreviations: ANOVA, Analysis of variance; CTA, conditioned taste aversion; CYP, cyclophosphamide; IMP, inosine 5=-monophosphate; MSG, monosodium glutamate; S⫹, stimulus associated with reinforcement; S-, stimulus associated with punishment; TSC, taste sensory cell.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.07.006

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2005). Although some of 4-hydroxy CYP or its tautomer, aldophosphamide, is converted by aldehyde dehydrogenase to carboxy phosphamide (a nontoxic compound), most is converted to two toxic byproducts, acrolein and phosphoramide mustard (Powers and Sladek, 1983). These compounds diffuse out of hepatic cells and reach various locations within the body through blood circulation. The cells in the S-phase of the cell cycle are more vulnerable to attack because DNA synthesis starts in that Sphase and the two strands of DNA are in the unwound state. These compounds diffuse through the cell and nuclear membranes and, after entering the nucleus, they attack DNA at the guanine N-7 position by forming intra- or interstrand crosslinkages (Povirk and Shuker, 1994; Dong et al., 1995). This causes the cell cycle to arrest and initiates DNA repair. Often the DNA damage is so intense that the cell’s intrinsic machinery cannot repair the damage and the cell undergoes apoptosis. The greatest tragedy of all chemotherapy drugs, including CYP, is that they cannot distinguish between cancerous cells and normal cells that are in their proliferative phase. This leads to the death of normal cells along with cancerous cells. Normal cells with a high turnover rate like the cells of hair follicles, cells lining the intestine, and so forth, are vulnerable to the attack by CYP, thereby leading to serious side effects such as loss of hair and nausea. Taste buds of mice are comprised of approximately 50 –100 cells of four morphologically distinct cells types, taste sensory cells (TSCs) types I–III and basal cells (also called type IV cells) (Kinnamon, 1987; Stone et al., 2002; Finger, 2005). TSCs are believed to have an average life span of 8 –12 days (Delay et al., 1986) and thus must be replaced continuously (Beidler and Smallman, 1965; Farbman, 1980). The cells responsible for the cell replacement cycle are thought to be located around the base of taste buds or in the adjacent basal epithelial cell layer, or the perigemmal cells located outside the taste buds (Beidler and Smallman, 1965; Delay et al., 1986; Cummings et al., 1987; Stone et al., 2002; Miura et al., 2006; Nakayama et al., 2008; Okubo et al., 2009). These basal cells are designated as progenitor cells which are believed to divide asymmetrically to produce transit amplifying cells (Farbman, 1980). In turn, the transit amplifying cells give rise to the specialized TSCs (Beidler and Smallman, 1965; Farbman, 1980; Delay et al., 1986; Miura et al., 2006; AsanoMiyoshi et al., 2008). With the current knowledge of CYP, it can be hypothesized that if CYP has a direct effect on the taste system that is not related to learned taste aversions, then it is most likely to have a detrimental effect on a component of the system with a high rate of cellular turnover such as the progenitor cells involved in the TSC replacement cycle or, more likely, the transit amplifier cells (daughters of progenitor cells) which have a higher rate of proliferation. We hypothesized that CYP causes disturbances in taste function by directly affecting taste buds and taste cells. As an initial test of the effects of CYP on taste functions, we focused on the prototypical umami substances, monosodium glutamate (MSG) and inosine 5=-monophos-

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phate (IMP). These substances were selected because in rodents, they elicit a complex taste involving several taste qualities. These qualities include a salt quality associated with the sodium ion and the unique umami taste qualities elicited by the glutamate anion of MSG. CTA experiments have shown that IMP elicits many of the same taste qualities as MSG (Yamamoto et al., 1991; Wifall et al., 2007). Even so, rats can readily discriminate between the two taste substances (Wifall et al., 2007), indicating that although the two substances share some taste qualities, they also possess taste qualities not shared by the other substance. In rodents, the addition of amiloride (a sodium channel blocker) to either MSG or IMP results in a potential sweet quality (by human standards) (Yamamoto et al., 1991; Chaudhari et al., 1996; Bachmanov et al., 2000; Bachmanov and Beauchamp, 2001; Stapleton et al., 2002; Heyer et al., 2003; Zhao et al., 2003). These findings suggest that a variety of taste receptors and TSCs are activated by these substances (Yasuo et al., 2008; Delay et al., 2009; Yoshida et al., 2009; Chaudhari and Roper, 2010). Besides their complex nature, MSG and IMP are often added to foods to enhance their palatability (Bellisle et al., 1991, 1996). This characteristic makes umami substances potentially useful as a taste enhancer for cancer patients. Schiffman et al. (2007) found that efforts to make foods more palatable can increase nutritional intake and increase quality of life over the course of treatment and beyond. It would be valuable to know whether umami taste is affected by CYP and whether umami could be an effective food enhancer for these patients. The effects of CYP were studied with behavioral tests that did not depend on CTA methods. We also analyzed changes in the taste epithelium by evaluating the morphology of taste buds and salivary function after injection of CYP. In the present study, we report an unexpected biphasic effect of CYP on umami taste discrimination and taste sensitivity which appears to be the result of druginduced disturbances in mouse taste epithelium.

EXPERIMENTAL PROCEDURES Subjects Male C57BL/6J mice, obtained from Jackson Laboratory (Bar Harbor, ME, USA), were housed in groups of 2– 4 mice. They were at least 3-months-old and weighed between 25 and 32 g at the start of the experiment. The mouse colony was maintained on a 12/12-h light/dark cycle with lights turned on at 7 AM and off at 7 PM. Purina Mouse Chow (Prolab RMH 3000) was available ad libitum. The mice in behavioral experiments were adapted to a 22-h water deprivation schedule beginning 1 week before the start of training. These mice were tested at the same time each day between 8 AM and 12 PM. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Vermont. All experiments were designed to minimize the number of mice required to test each experimental question.

Chemical reagents CYP (cyclophosphamide monohydrate, 97%) was obtained from Acros Organics (NJ, USA). MSG (monosodium L-glutamate) and IMP were obtained from Sigma (St. Louis, MO, USA) and Acros Organics (NJ, USA), respectively. All taste solutions were pre-

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pared daily with Millipore-filtered water (Millipore, Billerica, MA, USA) mixed with 100 ␮M amiloride (Sigma) to minimize the taste of sodium associated with either umami compound (Heck et al., 1984). Amiloride is not detectable by mice at this concentration and is capable of increasing the sodium taste threshold to ⬎500 mM (Eylam and Spector, 2003; Eylam et al., 2003).

Behavioral tests These experiments were designed to test whether there were any alterations of taste discrimination and detection thresholds after an i.p. injection of CYP. We used a taste discrimination method in which mice must distinguish between the qualitative characteristics of the tastes of two substances, MSG and IMP. A similar procedure was used to assess the effects of CYP on detection thresholds for these two substances. Apparatus. Following established methodology (Spector, 2003; Delay et al., 2006), discrimination and threshold experiments were carried out in five computer-controlled gustometers (Knosys Inc., Lutz, FL, USA; Brosvic and Slotnick, 1986) fitted in individual bench top stations. Each gustometer consisted of a Plexiglas operant chamber with dimensions of 17 cm long⫻12 cm wide⫻12 cm high. A small circular opening, 1 cm in diameter, was present in one of the walls at a height of 2 cm above the floor of chamber. A fan was mounted to the ceiling of the chamber for positive pressure airflow into the chamber. A piezo buzzer (Jameco Electronics, Belmont, CA, USA) was mounted in the ceiling of the chamber directly over the lick spout. When activated, the buzzer produced a 2.9 kHz continuous tone of 80 –90 dB inside the chamber. Taste solutions and water were delivered through a stainless steel lick spout, the tip of which was located in the center of the opening. The floor of the chamber had a removable stainless steel plate placed in such a way that mice had to stand on the plate to access the spout. Taste stimuli and water were stored in eight 3-ml and one 10-ml unpressurized syringe barrels, respectively, mounted on a Plexiglas rack located near the cage. The bottoms of the syringe barrels were 7.5 cm above the drinking spout. The barrels were connected by capillary tubing (Cole-Parmer Instrument Company, IL, USA) to individual stainless steel tubes inside the lick spout. The tip of each tube was recessed 2 mm from the end of spout. Fluid delivery was regulated by pinch valves (Parker Hannifin, Cleveland, OH, USA; P/N 0030507-900) designed to operate very quickly and quietly. A lick was counted each time the mouse’s tongue made contact with the lick spout, allowing a flow of 60 ␮A current through the stainless steel floor plate. Visual cues were minimized by mounting the syringe barrels on a stimulus rack facing away from the cage at least 30 cm from the lick spout. Olfactory cues were minimized by using fresh solutions each day, using small diameter delivery tubes recessed inside the lick spout, small sample sizes, and the chamber fan blowing air through the chamber and exiting through the lick spout opening. Auditory cues were masked by a louder independently operated solenoid (mounted 2.5 cm directly above the lick spout) that opened and closed simultaneously with one of the pinch valves, and by background masking noise generated by a Sleep machine (Radio Shack, Fort Worth, TX, USA) (SPL, A scale: 75⫾5 dB). General procedures. Discrimination and threshold methods were similar to those described in previous works of this laboratory (Delay et al., 2004, 2006). It is based on the principle that the mice can differentially respond to qualitative differences between two different taste substances. Each training or test session lasted 50 min or 160 trials, whichever happened first. A half hour after the end of the session, a water bottle was placed on the home cage for 30 min to maintain the 22 h deprivation schedule. To initiate a trial during the session, a mouse had to lick the spout an average of 20 times (VR-20: computer randomly selected a number within

a range of 5–35) to receive 3.5 ␮l water to rinse the palate and encourage further licking. This was followed by a 3-s delay after which the mouse again had to lick an average of 20 times to get 7 ␮l of the stimulus delivered. After licking the taste solution, the mouse had a 2-s decision period in which to decide whether the solution was an S⫹ or an S⫺ stimulus. Responding during the last 0.4 s of the decision period determined the response outcome. If the stimulus was the S⫹ and the mouse continued licking during the last 0.4 s of the decision period, a 10-␮l water reinforcer was delivered and a correct “detection” was registered. If the mouse missed the S⫹, indicated by licking during the 0.4 s period, the intertrial interval began immediately after the end of the decision period and an incorrect response (“miss”) was registered. If the stimulus was an S⫺ and the mouse continued to lick during the last 0.4 s of the decision period (“false alarm”), an aversive stimulus was presented (mild shock or a loud tone plus a time out). A correct response was also counted (“correct rejection”) when the stimulus was S⫺ and the mouse stopped licking during last 0.4 s of the decision period. This response prevented the presentation of the aversive stimulus (avoidance). An intertrial interval of 10 s occurred between each trial. Discrimination experiments. Three discrimination experiments were completed. In the first two experiments, an incorrect response to the S⫺ resulted in the presentation of shock. When shock was used, shock intensity was titrated for each mouse to just above the threshold to induce avoidance of the shock but not to stop licking completely. The mouse experienced the shock only if it licked the spout during shock presentation. Even though the intensity of shock (15–20 V) was minimally detectable by the mouse and was experienced only briefly if at all on any trial, it was possible that shock aggravated inflammation that might have been induced by CYP in the tongue epithelium. Therefore, the third discrimination experiment replicated all procedures but with a high intensity 4 s tone stimulus plus a 10 s timeout punisher to replace shock. After 3 weeks of training, the discrimination performance of all mice was consistently stable at ⬎85% accuracy. At this point, all mice were taken off water deprivation and 24 h later the mice randomly assigned to the CYP injection group received a single IP injection of CYP (75 mg/kg body weight), whereas the rest of the mice received an injection of the vehicle (0.9% saline). The mice were given a recovery period of 24 h and were housed in filtertopped cages with complete access to food and water. At the end of this period, they were returned to the deprivation schedule and behavioral testing resumed 22 h later. Half of the mice in each discrimination experiment were tested with MSG as the S⫹ stimulus, whereas half had IMP as the S⫹ stimulus. In the first two experiments, 60 mice were trained and tested with shock as the punisher. In the first experiment, 12 mice (six CYP-injected, six saline-injected) were tested for 16 days after injection to establish a timeline for the effects of CYP on taste functions. The remaining 48 mice were trained and tested in a similar manner in the second experiment except that mice were removed at specific time points for morphological analysis (described later in the text). In the third experiment, 10 mice (eight CYP-injected, two saline-injected) were trained and tested with the tone and timeout as punisher. An equal number of S⫹ and S⫺ trials were presented during each session and the order of S⫹/S⫺ presentations followed blocks of randomly selected Latin square sequences. To minimize concentration as a discrimination cue, four concentrations of MSG (25, 50, 100, and 150 mM) and IMP (2.5, 5, 10, and 15 mM) were presented each day. These concentrations were selected on the basis of published data (Delay et al., 2006; Wifall et al., 2007; Murata et al., 2009) and preliminary studies. Each stimulus solution was stored in a randomly assigned syringe barrel each day. The reinforcer tube always contained deionized water with amiloride.

N. Mukherjee and E. R. Delay / Neuroscience 192 (2011) 732–745 Detection threshold experiment. CYP injections resulted in disturbances in discrimination performance, raising questions about whether the drug might have elevated detection thresholds for MSG and IMP. Clinical reports suggest chemotherapy drugs may elevate taste thresholds (Hall et al., 1980; Berteretche et al., 2004). If so, the disturbance in discrimination may be the result of thresholds elevated to above the concentrations used in the discrimination experiments. This experiment addressed this possibility by measuring the thresholds for MSG and IMP after injections of CYP. The procedures of the threshold experiment were similar to the discrimination tests with the following modifications. Ten (six CYP-injected, four saline-injected) C57Bl/6J mice of the same description as those used for the discrimination experiment were used to determine the thresholds of IMP, and 10 (six CYP-injected, four saline-injected) mice were used to determine the threshold of MSG. All the animals were tested in the same operant chamber as described for the discrimination experiments. Each mouse was trained with water as S⫹ and either MSG or IMP as S⫺. A correct detection of the S⫹ delivered water reinforcer to encourage the licking of the sprout. A 4-s tone plus a 10-s timeout period occurred as a punisher after each incorrect response to the S⫺. During a test session, an animal was presented with six concentrations of S⫺, all in a randomized block order, but balanced so an equal number of S⫹ and S⫺ trials occurred during a 160-trial session. Each concentration was stored in a different barrel each day to minimize the possibility that the mice could identify a taste stimulus on the basis of location of stimulus delivery within the spout. The concentrations used for IMP were 0.001, 0.01, 0.1, 0.3, 1, and 3 mM. The concentrations used for MSG were 0.01, 0.1, 1, 3, 10, and 30 mM. Once performance appeared stable (approximately 4 weeks of training), three sessions were conducted to obtain a concentration–response function for each mouse with which to estimate the threshold of the assigned umami substance. After the third session, all animals were rehydrated and 24 h later the experimental group received an IP injection of CYP at the same dose as mentioned earlier, whereas control group received an injection of saline. After the recovery period of 48 h, threshold testing of mice in both groups resumed until 16 days after injection. Detection thresholds were monitored daily to determine if they were elevated to a level exceeding concentrations used in the test session and, although it was not necessary, to make adjustments in stimulus concentration to avoid loss of stimulus control.

Morphological analysis To study potential reasons behind the disruptions of taste function after CYP injections, the cellular morphology of taste papillae, taste buds, and salivary glands of the tongue were examined. As noted earlier in the text, animals used to evaluate taste papillae and taste buds were initially trained and tested on the discrimination test (experiment 2). This procedure allowed us to verify that the disruption seen in the original discrimination task was replicated in the mice being evaluated for morphological state of the taste buds and papillae. The mice were perfused at days 0 (no injection), 4, 8, 10, 12, and 16 after CYP injection. On the day of perfusion, the mice were first run on the discrimination task, and then rehydrated before perfusion. All perfusions were done with ice-cooled PBS followed by 4% paraformaldehyde in PBS (Electron Microscopy Sciences, PA, USA) and the tongues were removed for analysis. To determine whether the numbers of fungiform papilla were affected by CYP, fungiform papillae were counted with attention toward the presence or absence of pores. The number of functional and nonfunctional papilla in control and treated animals can provide an initial indication of changes in the lingual epithelium in response to CYP. Each fungiform papilla contains at least one potential taste bud which may be in a functional or nonfunctional

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state, indicated by the presence of a taste pore (Iwasaki et al., 1997; Parks and Whitehead, 1998). After fixation, whole tongues were stained with Fast Green and examined under a dissecting microscope (Nikon SMZ1000) (Avon, MA, USA) equipped with a Photometrics Cool Snap EZ camera (Tucson, AZ, USA). Staining with Fast Green enabled two observers, blind to the injection condition, to view the papilla and differentiate the papilla with and without a pore. To relate the data obtained from behavioral experiments with the morphology analysis, mice were taken out of discrimination experiment 2 at 0, 4, 8, 10, 12, and 16 days postinjection. To refine our understanding of the morphological changes better, days 2 and 6 were later added to the time points. A total of eight tongues per day were evaluated for fungiform papilla with and without pores for days 0, 4, 8, 10, 12, and 16 postinjection and six tongues per day were evaluated for days 2 and 6. Following this, each tongue was cryoprotected with a 30% sucrose solution and frozen-sectioned at thickness of 20 ␮m. Every third section was selected from the anterior 1 mm of the tongue and then stained with Hematoxylin and Eosin (Ricca Chemical Company, Arlington, TX, USA) to look at the morphology of the tissue. For the purpose of staining, the slides were hydrated briefly with distilled water followed by Hematoxylin staining for 6 min. The excess stain was washed out under running tap water for 8 min followed by distilled water rinse for 5 min. The slides were then stained with Eosin briefly and cover-slipped using Permount (Fisher Scientific SP15-500). The images were captured using either a Zeiss Axioskop2 with a Photometric Cool SNAP EZ camera or a Nikon Eclipse E600 Scope fitted with a color camera (Spot RT KE Diagnostic Instruments Inc.) and Spot acquisition (Spot Advanced, version 4.6) software. Every third section from the caudal part of the tongue starting from anterior foliate papillae was stained to examine the morphology of circumvallate taste buds and Von Ebner glands. ImageJ (NIH, Bethesda, MD, USA) was used to evaluate the height and width of Von Ebner glands, fungiform taste buds, and circumvallate taste buds. To measure the height of a taste bud, a line was drawn along the longitudinal axis from the apical to the basal end of the taste bud. Another line was drawn perpendicular to the longitudinal axis at the point where the width was maximal. To measure the area of the Von Ebner glands, an ROI outline was drawn and the area was calculated by ImageJ.

Salivary function Salivary gland functioning is important for proper functioning of taste buds and reduced salivary secretion has been shown to reduce taste sensitivity (Matsuo et al., 1997; Matsuo, 2000). Previous oncology research has reported that xerostomia often occurs after radiation or chemotherapy treatment of cancer patients (Huguenin et al., 1999; Wickham et al., 1999; Cheng and Chang, 2003; Trotti et al., 2003; Shiboski et al., 2007; Cheng, 2007), presumably from damage to salivary glands and reduced salivary output. Thus, the food may not be diluted properly and may not reach the receptors (Matsuo, 2000; Mese and Matsuo, 2007). Even though the taste stimuli in the behavioral experiments were in solution, we wanted to determine whether reduced salivation could be partially responsible for the deficits in taste functions. For this assessment, we adapted the method originally described by Lin et al. (2001). The experimental group (n⫽5) received a 75 mg/kg i.p. injection of CYP, whereas the control group (n⫽5) received an equal volume injection of isotonic saline. Saliva was collected from these mice before (day 0) and on days 4, 8, 10, 12, and 16 after injection. On these days, each mouse was given an i.p. injection of the muscarinic acetylcholine receptor agonist, pilocarpine (1.25 ␮g/g body weight; MP Biomedicals, LLC). After the injection of pilocarpine, the mouse was placed in a dry cage without bedding. The first collection was done by a person blind to the test conditions 5 min after the injection of pilocarpine or saline. To collect a sample of saliva, each mouse was held in a restrained

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upright position till the saliva was collected by inserting the pipette tip inside the mouth and moving it horizontally across the tongue for approximately 1 s. Saliva was collected three times during each test session at an interval of 5 min between each collection. The weight of saliva collected from each mouse was measured and the volume was calculated (Lin et al., 2001). The advantage of sampling salivary secretion with this method is that saliva can be collected from the same mouse at multiple time points without anesthesia or removal of salivary glands.

Statistical analysis All the statistical analyses were done with the SPSS software (IBM SPSS Statistics, version 19, IBM Corporation, Chicago, IL, USA) and Graphpad Prism 5 software (Graphpad software Inc., La Jolla, CA, USA). Analysis of variance (ANOVA) procedures were used to evaluate the effects of drug condition (two levels, CYP and saline), treated as a between subject variable, and taste substance (two levels, MSG and IMP) and days (17 levels, two preand 15 postinjection) treated as within subject variables on behavioral and salivary measures. Initial ANOVAs did not detect any significant differences in the performance of animals related to detection of S⫹ or S⫺ assignment nor to the specific taste substance (MSG vs. IMP). Therefore, subsequent analyses examined the percent correct detection score for each pair of MSG and IMP stimuli with the same ordinal rank in their respective concentration gradient. The correct detection score of each mouse on each day was calculated as follows (Delay et al., 2006): % Correct Detection Score ⫽ [(% Detection of S⫹) ⫹ (% Detection of S⫺)]/2. This method has the advantage of avoiding possible between subject differences in motivational states or response strategies that might have been used by individual mice (Heyer et al., 2004). A correct detection score of 50% was treated as chance performance. When the injection group by day interaction was significant in the following ANOVAs, the correct detection scores of the mice of both groups were subjected to post hoc tests to determine the days in which the performance of the two groups were significantly different.

RESULTS In general, the results of all of these experiments indicated that there is a two-phase disturbance in taste observed after a single injection of CYP. The first phase was observed as an immediate drop in performance at 2– 4 days after injection and the second phase was observed as another drop in performance in a period that began 8 –9 days after injection and ended about 12–13 days after injection. Discrimination experiments Experiment 1. This experiment was designed to determine whether the discrimination performance of the mice was affected by CYP (Fig. 1). A mixed model repeated measure ANOVA identified significant effects of injection group [F(1,10)⫽270.74, P⬍0.0001], days [F(16,160)⫽30.86, P⬍0.001], and group by days interaction [F(16,160)⫽31.71, P⬍0.001]. A follow-up ANOVA on only the preinjection data indicated that the detection rates of the control and CYP-injected mice did not differ significantly before drug injection. An ANOVA performed on the postinjection data indicated significant differences between the control and the CYP-injected groups across days. Post hoc analysis with simple effect tests revealed

Fig. 1. Comparison of correct detection rate of MSG and IMP made by mice before and after CYP or saline injection when mild shock was used as a punisher. The graph shows the mean (⫾ SEM) percentage of correct detections (Y-axis) over days (X-axis). The performance of both groups was similar before the injection (days ⫺2, ⫺1). On day 0, the experimental group (dash line) received the CYP injection and control group (solid line) received the saline vehicle. On days 2– 4 after injection, performance of CYP-injected mice dropped to near chance levels (P⬍0.001) and then gradually improved to control levels. A second drop in the performance by CYP-injected mice was observed on days 9 –12 postinjection (*** P⬍0.001).

that there was a significant drop in performance of the CYP group compared with the control group on days 2, 3, and 4 [F(1,135)ⱖ32.36, Pⱕ0.001]. In addition, the performance of the CYP group was significantly poorer on days 9 –12 postinjection [F(1,135)ⱖ40.67, Pⱕ0.001]. Experiment 2. The purpose of this experiment was to determine whether the animals used for morphological analysis were behaviorally comparable with the corresponding drug conditions at the same interval in experiment 1 as at postinjection days 0 (no injection), 4, 8, 10, 12, and 16. For example, separate ANOVAs were conducted to compare the performance of the mice removed 4 days after injection for morphological analysis with the experiment 1 CYP group (experiment 1 day 4 vs. experiment 2 day 4). Even though there were significant changes in performance over days detected in all CYP-injected groups, none of these analyses found a significant group difference [Fⱕ0.87] or a group by days interaction [Fⱕ0.34] related to the two discrimination experiments. In essence, the CYP-injected mice in experiment 2 replicated the data observed in experiment 1. Experiment 3. In the final discrimination experiment, mild shock was replaced with a noise⫹timeout combination as a punisher to rule out any physiological changes that might result from aggravation due to the mild shock (Fig. 2). The mixed model repeated measure ANOVA indicated significant main effects of injection group [F(1,8)⫽258.54, P⬍0.001], days [F(16,128)⫽2.69, P⬍ 0.001], and group by days interactions [F(16,73)⫽3.04, P⬍0.001]. A follow-up ANOVA on only the preinjection data indicated that the detection rate of the control and CYP-injected mice did not differ significantly before drug injection. An ANOVA performed on the postinjection data

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Fig. 2. Comparison of correct detection rate of MSG and IMP made by mice before and after CYP or saline injection when noise⫹timeout was used as a punisher. The graph shows the mean (⫾ SEM) percentage of correct detections (Y-axis) over days (X-axis). The performance of both groups was similar before the injection (days ⫺2, ⫺1). As in the previous experiment, CYP and saline injections were given on day 0. Performance of CYP-injected mice (dash line) dropped to near chance levels on days 2– 4 after injection (P⬍0.001), and then returned to control levels (solid line). A second drop in the performance by CYPinjected mice occurred on days 9 –12 postinjection (*** P⬍0.001).

indicated that there were significant differences between the control and the CYP-injected groups across days (P⬍0.001). Simple effect tests revealed that there was a significant decline in performance of the CYP group compared with the control group on days 2 and 3, and 9 –12 postinjection [F(1,135)ⱖ11.82, P⬍0.001]. Because the number of subjects (n⫽2) for the control group was small for the aforementioned experiment, a repeated measure ANOVA was used to compare the experiment 1 control group with this control group. The results indicated that there was no significant difference between these two control groups (all Ps ⬎0.05). An additional ANOVA was performed in which the control groups of experiments 1 and 3 were combined to obtain a total of n⫽8 for the control group to compare with the CYP-injected group of experiment 3 (n⫽8). This ANOVA indicated significant main effects of injection group [F(1,14)⫽474.56, P⬍0.001], days [F(16,224)⫽9.23, P⬍0.001], and group by days interactions [F(16,224)⫽10.23, P⬍0.001]. Simple effect tests revealed that there was a significant drop in performance of the CYP group compared with the control group on days 2 and 3 (Pⱕ0.001). In addition, the performance of the CYP group was significantly poorer on days 9 –12 postinjection (Pⱕ0.001), thereby verifying the original findings with the small control group and replicating the findings of experiment 1. Water bottles were inadvertently left on the cages for longer than the normal 30-min period after the day 14 session, causing a dip in performance in both groups on days 15 and 16. To determine whether there might be shifts in motivational states because of CYP, repeated measure ANOVAs were used to compare the body weights and the number of trials completed by the saline and CYP-injected mice each day over the course of experiment 1. No significant group differences were detected in body weights across days.

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However, a significant difference between the control and CYP group in terms of number of trials performed over days was detected [F(17,238)⫽3.83, P⬍0.001], so the data were partitioned for further analysis. No difference in the number of trials/day between the control and CYP group during the preinjection period were detected (Bonferroni-corrected t-tests). For example, control mice completed an average of 104 trials before their injection, 109 trials on day 7 and 115 trials on day 16. CYP-injected mice completed an average of 103, 105, and 112 trials on the same days, respectively. There was, however, a significant drop in the number of trials completed by the CYP-injected group (mean⫾SEM ⫽ 54.13⫾8.18) compared with the saline-injected group (100.25⫾5.50) on day 2 only (P⬍0.01) after injection. Thus, a relatively short duration disturbance in motivation may have affected the performance of the standard CYP-injected mice during the first session after the injection, but is unlikely to have a strong impact in subsequent sessions. Detection threshold experiments The threshold experiments were designed to see if there is a shift in taste sensitivity after administration of CYP. The detection threshold for the assigned S⫺ (MSG or IMP), defined as the concentration detected 50% of the time, was determined each day for each mouse, assuming a logarithmic scale of stimulus concentrations. MSG thresholds. The mean thresholds for MSG of saline- and CYP-injected groups over days are shown in Fig. 3. No group differences in MSG threshold were observed during the preinjection period. However, in the

Fig. 3. Detection thresholds of MSG before and after CYP or saline injection. Detection thresholds, a measure of taste sensitivity, were defined as the concentration of MSG detected 50% of the time. The graph shows the geometric mean (⫾ SEM) threshold concentration of MSG (Y-axis, log scale) across days (X-axis) postinjection. Days ⫺1, ⫺2 indicate thresholds before the injection. On day 0, mice were injected with either CYP (dash line) or saline (solid line). CYP-injected mice showed significant elevations of MSG detection thresholds on days 2, 3, and 8 –12 after injection (** P⬍0.01, *** P⬍0.001). A Mann–Whitney test indicated that CYP significantly elevated MSG thresholds on day 13 as well (⫹ P⬍0.005).

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Fig. 4. Detection thresholds of IMP before and after CYP or saline injection. Detection thresholds were defined as the concentration of IMP detected 50% of the time. The graph shows the geometric mean (⫾ SEM) threshold concentration of IMP (Y-axis, log scale) across days (X-axis) postinjection. On day 0, mice received either a CYP (dash line) or saline (solid line) injections. CYP significantly elevated IMP detection thresholds on day 2, 3, and 10 –14 after injection (** P⬍0.01, *** P⬍0.001). Mann–Whitney tests indicated that CYP also significantly elevated thresholds on days 4 and 9 (⫹ P⬍ 0.005).

postinjection period, there was a significant main effect of days [F(16,128)⫽10.48, P⬍0.001], injection group [F(1,8)⫽42.09, P⬍0.001], and a significant group by days interaction [F(16,128)⫽10.71, P⬍0.001]. Simple effects tests revealed that there was a significant elevation in the MSG detection threshold of the CYP-injected mice on days 2 and 3 (Ps ⬍0.001), and days 8 –12 postinjection (Ps ⬍0.01). For example, thresholds for MSG on day 2 postinjection averaged 4.341⫾0.491 mM (geometric mean⫾SEM) for the CYP-injected mice, whereas the thresholds for the saline-injected mice averaged 0.773⫾0.042 mM. The greatest group difference in MSG thresholds was seen on postinjection day 12. Thresholds of CYP-injected mice averaged 6.783⫾1.064 mM, whereas the MSG thresholds of the saline-injected mice averaged 0.313⫾0.054 mM. MSG thresholds on day 13 did not reach significance (P⫽0.057) with parametric testing, probably because of the small sample sizes. However, since there was no overlap between the distributions of thresholds of the CYP-injected and saline-injected groups (Fig. 3), a Mann–Whitney test was applied. This test indicated that all of the thresholds of the CYP-group were significantly higher than the thresholds of the saline-injected group on day 13 (P⬍0.005). IMP thresholds. The mean thresholds for IMP of the two drug groups over days are shown in Fig. 4. There were no group differences in thresholds during the preinjection period. However, there were significant effects of days [F(16,128)⫽7.81, P⬍0.001], injection group [F(1,8)⫽ 97.27, P⬍0.001], and group by days interaction [F(16,128)⫽8.21, P⬍0.001] on the thresholds for IMP in the postinjection period. Simple effects tests indicated that after CYP injection, there was a significant increase in IMP

detection threshold on day 2 (P⬍0.001) and day 3 (P⬍0.01) after CYP injection. The largest drug-induced elevation of IMP threshold was detected immediately after injection. On day 2, the IMP threshold of CYP-injected group was 2.197⫾0.445 mM, whereas the average threshold of control mice was 0.021 mM⫾0.0045 on the same day. In addition, there was a second significant elevation in IMP thresholds on days 10 –14 after injection [Fs(1,135) ⱖ13.51, Ps ⬍0.001]. On day 12, the average IMP threshold for the CYP mice was 1.852⫾0.254 mM compared with an average threshold of 0.004⫾0.0003 mM for control mice. Parametric testing of the threshold data for days 4 and 9 did not reveal any differences between the two groups. However, there was no overlap of the distributions of thresholds for the CYP-injected and saline-injected groups. Mann–Whitney testing of these data indicated that all of the thresholds of the CYP-group were significantly higher than the thresholds of the saline-injected group on days 4 and 9 (P⬍0.005). In general, the analyses of the threshold data indicated that there are two periods after injection of CYP when mice showed a loss of sensitivity (increased thresholds) for IMP and MSG. The first period coincides with the first deficits in discrimination, whereas the second period of reduced sensitivity bracketed the second period of reduced discrimination performance. However, these elevations in thresholds for MSG and IMP were not enough to account for the discrimination deficits. Papillae assays Because the behavioral data revealed two periods of taste deficits after CYP administration, the lingual epithelium was examined for potential changes that might correspond to the disruptions in taste function. The ANOVA of the fungiform taste papillae counts indicated that by day 2 after CYP injection, the number of fungiform papillae decreased sharply, and this number did not recover until about day 12 after injection (Fig. 5). The one-way ANOVA comparing the number of papilla in control (day 0) and CYP-injected groups (days 2, 4, 6, 8, 10, 12, and 16) indicated statistically significant differences between the groups [F(7,59)⫽ 26.07, P⬍0.001]. Post hoc comparisons (Dunnett’s test) using day 0 as the control group indicated that the number of papillae on the tongues of the control group was significantly greater than the rest of the groups [all Ps ⬍0.01] except on days 12 and 16. The number of taste papilla with a taste pore also dropped by about half and did not recover until 16 days after CYP injection [F(7,59)⫽50.49, P⬍0.001]. Dunnett’s test indicated a significant drop in the number of fungiform taste papilla with pores in all groups compared with control mice (day 0) except the day 16 group (all Ps ⬍0.01). In general, papillae with pores decreased over days, reaching the lowest number 10 days postinjection. To examine further CYP-induced disruptions in taste buds, the harvested tongues were sectioned for Hematoxylin and Eosin staining to examine the fungiform and the vallate taste buds. Not only were the number of fungiform taste buds decreased but the organization of cells inside

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Fig. 5. Mean (⫾ SEM) number of fungiform papillae and fungiform papillae with a taste pore across days postinjection. CYP significantly decreased the number of fungiform taste papillae on postinjection days 2, 4, 6, 8, and 10 compared with noninjected control mice (day 0). There was also a significant drop in the number of fungiform taste papillae with pores on days 4, 6, 8, 10 and 12 compared with day 0 (** P⬍0.01).

the remaining taste buds appeared to be affected and did not seem to recover until about 12 days after injection (Fig. 6A–E). The general size of fungiform papilla was studied using height and width as the parameters. One-way ANOVAs revealed a difference in the height [F(5,27)⫽12.57,

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P⬍0.001] but not the width of papillae between salineinjected control mice (day 0) and CYP-injected groups (days 4, 8, 10, 12, and 16). Dunnett’s tests indicated that the height of taste buds only on day 4 was significantly reduced compared with day 0 (P⬍0.05). The effects of CYP on circumvallate papillae were not detectable as early as effects of the drug on fungiform papillae (Fig. 7). Nevertheless, the number of vallate papillae showed a drop over days [F(5,47)⫽24.97, P⬍0.001]. Dunnett’s tests indicated that on days 8, 10, and 12, the number of taste buds was significantly lower compared with day 0 (all Ps ⬍0.01), but by day 16, the number of taste buds recovered to the same levels as controls. These data suggest that in contrast to fungiform papillae, the number of circumvallate taste buds did not appear to decrease until 8 days after injection. Comparisons of Hematoxylin and Eosin-stained sections of circumvallate papilla (Fig. 6F–J) did not reveal any observable CYP-induced change in the shape of taste buds or the spindle-shaped taste cells on day 4. On day 8, the perigemmal cells forming the outer boundary of the taste bud appeared similar to the control (non–CYP-injected) taste buds. Also, taste cells within the buds appeared to be elongated, tapered toward each end, and extending from the apex to the base of the bud, but they lacked the appearance of being “packed” in the bud. Instead, there were vacant spaces between cells which ran parallel to the long axis of the taste bud. There were no

Fig. 6. Representative sections of fungiform (Panels A–E) and circumvallate (Panels F–J) taste buds on pre- and postinjection days. The upper row (Panels A–E) of the figure shows the images of fungiform taste buds and the lower row (Panels F-J) shows the images of circumvallate taste buds. The columns from left to right are images of taste buds from no injection control mice (day 0) (Panels A, F) and from CYP-injected mice 4 d (Panels B, G), 8 d (Panels C, H), 10 d (Panels D, I) and 12 d (Panels E, J) after injection. In (Panels A–E) fungiform taste buds appeared to be disturbed at 4 d (B) after CYP injection and continued to remain disrupted at 8 (C) and 10 d (D) after injection. They appeared to be recovering by day 12 (E). In (Panels F–J), the circumvallate taste buds of the control mice (F) and the day 4 mice (G) appeared morphologically comparable, but at days 8 (H) and 10 (I) postinjection, vacant spaces were observed inside the taste buds, possibly resulting from vacating cells without replacement. These spaces were not observed 12 d after injection (J). Scale bars⫽50 ␮m.

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Fig. 7. Mean (⫾ SEM) number of circumvallate taste buds across days postinjection. CYP significantly decreased the number of circumvallate taste buds on postinjection days 8, 10, and 12 compared with noninjected control mice (day 0) (** P⬍0.01).

detectable differences in the width of these taste buds but there was a detectable group difference in their height [F(5,47)⫽3.88, P⬍0.05]. Dunnett’s tests indicated that on day 8, the height of the circumvallate buds of the CYPinjected mice were significantly less than the height of the vallate taste buds of the day 0 group (P⬍0.05). Von Ebner gland assay Histological assay. We also evaluated sections of Von Ebner glands stained with Hematoxylin and Eosin to see whether these were affected by CYP. The height, width, and area of the gland were measured using ImageJ. One-way ANOVAs indicated a difference in the height [F(5,40)⫽17.87, P⬍0.001], and width [F(5,40)⫽22.72, P⬍0.001], and area [F(5,40)⫽10.91, P⬍0.001] of the gland between the control and CYP-injected groups. Post hoc testing indicated that the height, width, and area of the glands were significantly reduced on day 4 (Ps ⬍0.05) after injection, but had returned to normal dimensions on days 8, 10, and 12 after injection. Salivary function. Because the morphology of Von Ebner glands appeared to be affected by CYP immediately after injection, one might expect that the function of these glands is also affected. The ANOVA of the pilocarpine test results indicated significant injection group [F(1,8)⫽20.63, P⫽0.0019], days [F(5,40)⫽8.38, P⬍0.0001], and group by days interaction [F(5,40)⫽8.38, P⬍0.0001] effects on salivary output (Fig. 8). Bonferroni-corrected t-tests verified that saliva output of the CYP group was significantly less at 4 days after injection compared with the control group (P⬍0.001). However, the CYP-injected groups did not differ from the control group at any other time point.

DISCUSSION Previous research has attributed the detrimental effects of chemotherapy drugs on taste to the development of

learned taste aversions. This study was undertaken to see whether CYP, a widely used chemotherapy drug, had a direct effect on taste epithelium that corresponded to changes in taste specific behaviors. Our behavioral results revealed two separate periods of disturbance in umami taste in response to a single injection of CYP. The histological studies identified CYP-induced changes in taste buds and Von Ebner glands that correspond to the behavioral results, suggesting that CYP-induced disturbances in taste function have, at least in part, a biological basis. Previous studies have explained the CYP-induced detrimental effects on taste and food intake with the help of learning theory and drug-induced cognitive changes (Bernstein, 1978; Bernstein and Webster, 1980; Ahles and Saykin, 2002). CYP is considered to be a highly emetogenic drug as per the National Comprehensive Cancer Network guidelines (Viale, 2006). One of the immediate side effects of CYP is gastrointestinal disorder and emesis which leads to chemotherapy-induced nausea and vomiting. Other side effects include oral mucositis and xerostomia (dry mouth) which can contribute to changes in taste sensation (Cheng, 2007). According to learning theory, the pairing of the aforementioned adverse side effects of CYP with the tastes of different foods causes a negative association between food and sickness. This leads to the development of unintended CTAs which alter the hedonic values of taste substances. CTAs generalize readily to other similar tasting substances and thus, in the case of chemotherapy drugs, can result in substantial disruption of ingestive behavior and can cause avoidance of even highly preferred food (Bovbjerg et al., 1992). For example, one study (Bernstein, 1978) reported that children undergoing chemotherapy developed a strong aversion for food (novel flavor of ice cream) after pairing with CYP, resulting in subsequent avoidance of the taste; that is, they learned a CTA. These

Fig. 8. Mean (⫾ SEM) volume of salivary output induced by pilocarpine across days after injection. The graph shows the volume of salivary output (Y-axis) induced by pilocarpine (an ACh agonist) collected for saline-injected (solid line) and CYP-injected (dashed line) mice when tested at days 0, 4, 8, 12, and 16 d postinjection (X-axis). The volume of salivary output of the CYP group was significantly less on day 4 postinjection than saline-injected mice but not at any other time (*** P⬍0.001).

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studies also suggest that CTAs become more intense when coupled with multiple sessions of chemotherapy (Greene and Seime, 1987; Olin, 2001; Jansen et al., 2005). However, our results indicate that the deficits in taste functions observed in these experiments are not entirely because of the development of CTAs but are also because of the cytotoxic effects of CYP and possible disruptions in the taste cell replacement cycle. In the present study, the behavioral experiments were conducted to determine whether administration of CYP can compromise nonhedonic taste sensory functions such as taste acuity (discrimination) and/or taste sensitivity (detection thresholds) of mice. Two substances with quite similar umami taste qualities, MSG and IMP, were chosen for their complex taste qualities. The results of this study show that C57BL/6J mice, like rats (Wifall et al., 2007), can accurately discriminate between these two umami substances, suggesting that MSG and IMP also elicit taste qualities which make them distinguishable. Because the thresholds for these substances are different, as shown by the control groups in the threshold experiments, it was important to identify concentrations of each substance that elicited comparable behavioral effects to minimize the possibility that mice could use intensity as a discriminative cue. The comparable correct detection rates for MSG and IMP in the discrimination experiments argue that this was accomplished. It was also possible that the differences in the sodium components of MSG and IMP (a disodium salt) might contribute to the discrimination process. To minimize this possibility, amiloride, an ENaC antagonist, which can raise detection threshold for sodium to as high as 500 mM in mice (Eylam and Spector, 2003), was added to all solutions. However, amiloride does not block all sodium channels, and thus, may not have eliminated all of the sodium taste, especially for the highest concentrations of MSG. Even with these caveats, the results of the behavioral experiments revealed clear CYP-induced deficits. The results of all three taste discrimination experiments and the threshold experiments revealed a nearly complete loss of ability to discriminate between the two umami substances for at least 4 days immediately after drug administration, followed by a return to near normal performance on days 5– 8, and then a second decline in discrimination ability 9 –12 days after injection. In our experimental design, the onset of CYP-induced nausea was deliberately isolated from exposure to the taste stimuli to minimize any temporal contiguity between nausea and a taste stimulus needed to develop a learned aversion. Second, the temporal pattern of taste alterations observed in the behavioral data are not consistent with the effects of a CTA. The CTA literature (i.e. Spector, 2003; Bouton, 2007; Davis and Riley, 2010) as well as our laboratory’s extensive experience (Chaudhari et al., 1996; Stapleton et al., 1999; Heyer et al., 2003; Wifall et al., 2007; Eddy et al., 2009; Eschle et al., 2009) indicate that, even though a CTA can be extinguished, it does not oscillate over days or sessions without additional conditioning. Thus, our behavioral data suggest that the two-phase disturbances (especially the second phase) in taste functions after a single

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dose of CYP are unlikely, solely because of learning. Although the loss of sensitivity in these mice for both umami substances after administration of CYP is consistent with clinical reports of loss of taste sensitivity for sweet and salt tastes in chemotherapy patients (Bernhardson et al., 2008, 2009), the elevations in thresholds were not enough to prevent mice from detecting most or all of the concentrations of IMP and MSG in the discrimination experiments. Instead, these data, along with the changes detected in taste buds, suggest that CYP also has direct effects on taste epithelium that disrupt taste. The deficits in taste functioning observed 2– 4 days after injection may be in part due to cytotoxic effects of the drug. One immediate, but relatively short-term effect of CYP was a significant reduction in the number of trials performed by CYP-injected mice in the first discrimination experiment on day 2 after injection. These data suggest a relatively brief drug-induced change in appetitive motivation, possibly because of stomach malaise previously reported in CTA studies (Goodman and Gilman, 1975). Another immediate effect of CYP was the shrinkage of Von Ebner glands and the reduced salivary secretions detected 4 days after injection but not at later time points. Thus, the salivary glands also seem to be affected by the cytotoxicity of the drug and reduced salivation due to shrunken Von Ebner glands may have contributed to taste dysfunction in the first phase. An immediate but longer-term effect of CYP on taste tissues was the decrease in fungiform papillae, with and without a pore, to nearly half of control mice within 48 h after the injection, which remained at that level until 12 days after injection. The histological examination of remaining fungiform papillae revealed apparent disorganization of fungiform taste buds 4 days after injection. These findings suggest that the fungiform papillae were adversely affected immediately after the injection and were slow to recover. Recovery of these taste buds may have been related to the effects of CYP on keratinocytes surrounding the taste buds. The drop in the number of fungiform papilla including those with an open pore suggests that the keratinocytes forming the taste pore might also have been affected immediately by the drug. These keratinocytes have a relatively short life span of 5–7 days and, like TSCs, have a high turnover rate (Hume and Potten, 1976; Okubo et al., 2009), thereby making them vulnerable targets of CYP. A loss of the keratinocyte layer could have contributed to the loss of pore and disorganized fungiform taste buds. However, the effects of CYP on keratinocytes and whether these effects alter the integrity of taste buds need further exploration. Nevertheless, when these findings are taken together, the first loss in taste discrimination and sensitivity is likely due to a combination of effects of cellular toxicity on fungiform papillae and Von Ebner glands. The second disruption in umami taste function may have been related to the state of fungiform taste buds and to an apparent decline in the integrity of circumvallate papillae 8 –12 days after injection. It is possible that after the mice recovered from the cytotoxic effects of CYP, they were able to perform their behavioral tasks using the taste signals from the circumvallate taste buds. Circumvallate

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papillae exhibited no obvious changes until 8 days after injection, at which point there was a significant drop in number of taste buds along with apparent changes within the taste buds. At day 8, the perigemmal cells lining the outer boundary of circumvallate taste buds observed in the CYP-injected mice appeared to be similar to the control mice. However, within the taste buds elongated, cellshaped spaces were detected, suggesting that taste cells were “missing.” These spaces do not appear to be artifacts of processing as they were not observed in taste buds of control mice. It is possible that these spaces may be the result of aging TSCs dying and sloughing out of the taste bud without normal cell replacement. This speculation is suggested by the presence of morphologically intact circumvallate taste buds, presumably populated by postmitotic TSCs less susceptible to CYP on day 4, but frequent observance of the elongated spaces in the circumvallate taste buds at later time points suggests that there might have been a disruption in taste cell replacement cycle in response to CYP treatment. If CYP adversely affects the progenitor cells involved in the taste cell replacement cycle, for example, for at least 4 –5 days or longer after CYP injection, then mature TSCs for these taste buds may not be available during the second period of diminished taste capacity seen in the behavioral experiments. That is, the population of functional TSCs would be at a low point around 8 –12 days after CYP injection, approximately the same time frame in which deficits in taste discrimination and elevated thresholds were detected. By day 12, however, fungiform and circumvallate taste buds appeared to be returning to normal. However, further experiments designed to specifically look at the basal cell layer using BrdU or other markers of cell proliferation are needed to test this hypothesis. Curiously, fungiform taste buds seemed to be affected earlier than the circumvallate. It is possible that CYP might have entered the mouth with crevicular fluid. This fluid originates in the plasma and come to the mouth during chewing (Bartoshuk, 1990; Comeau et al., 2001). The fluid might contain the remnants of alkylating by-products of CYP. Another potential pathway by which these by-products might reach the oral cavity is through the gastroesophageal reflex (Bartoshuk, 1990). Because the fungiform taste buds are more on the surface of the tongue compared with circumvallate taste buds, they might be more vulnerable to the effect of the drug. The present experiments do not provide any insights into this differential effect. The reasons for this interesting finding are currently being explored. A critical question is whether other basic tastes also are similarly affected by CYP. It is tempting to say yes because one would expect all type II TSCs to be similarly affected by a disturbance of the cell replacement cycle. However, this needs to be addressed experimentally, especially because the clinical literature suggests that sensitivity for bitter substances actually increases in some patients during chemotherapy (Carson and Gormican, 1977; Hall et al., 1980). We are currently exploring this question further.

Interestingly, other chemotherapy drugs appear to have similar effects on intestinal stem/progenitor cells (Dekaney et al., 2009). Cell turnover in the intestine is much more rapid than in the taste bud, 1– 4 days, depending on the cell population involved. If the transit amplifying and/or progenitor cells are adversely affected by the chemotherapy drug, then the replacement cell cycles in the intestine are disrupted and the resulting effects over time may mimic the pattern seen in this study, with the temporal pattern adjusted to the life span of the cell being replaced. For example, the effects of the anticancer drug, cisplatin, on signaling within the common hepatic branch of the vagal nerve may be related to a disturbance in cell replacement cycle of the small intestine. Cisplatin is a platinum containing anti-cancer drug that induces apoptosis by DNA damage and like CYP, it falls into the category of emnetic drugs, causing nausea and anorexia. de Jonghe and Horn (2008) reported that in rats cisplatin induced pica at three points after injection: 1–2, 4, and 6 –10 days postinjection. Vagotomy alleviated most of the drug-induced pica effects, suggesting that cisplatin toxicity acts on enteroendocrine cells of the duodenum. Like the data reported in this study, the tri-phasic temporal pattern of drug-induced pica described by de Jonghe and Horn (2008) suggests that a single application of a chemotherapy drug may result in multiple points of functional disturbances over time after drug administration. These data also strongly identifies a need to examine the effects of chemotherapy drugs on cell replacement cycles of normal tissues when the drugs are administered multiple times and at various doses.

CONCLUSION In summary, these results indicate that CTA is not the only possible explanation of CYP-induced taste alterations. The findings reported in this study clearly show that the drug also has detrimental effects on taste epithelium of the tongue. Our two behavioral tests showed that a single dose of CYP can lead to a two-phase disturbance in the nonhedonic taste functions like taste acuity and taste sensitivity of umami substances. The first phase of taste disturbance (2– 4 days postinjection) can be attributed to the cytotoxicity of the drug exerting its effect on fungiform taste buds, Von Ebner glands, and motivational deficits of the mice because of possible stomach malaise. The second phase of taste disturbance (9 –12 days after injection) can be explained by the alteration in taste cell replacement cycle which inhibits the timely replacement of aging cells. These results are consistent with the effects seen in circumvallate sections of tongue after radiation therapy (Yamazaki et al., 2010) where they observed similar depletion of basal layer at 4 days after irradiation and repopulated basal layer by 8 days after radiation therapy with 15 Gy X-ray. Their behavioral data, acquired by two bottle preference testing, indicated an alteration of taste between days 4 –20 after irradiation. Shi et al. (2004) and Yamashita et al. (2009) have also reported umami taste dysfunction in patients receiving radiation therapy. Further research designed to see detailed effects of the drug on

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progenitor cell lines, mature cells, and nerve fibers will be able to evaluate the severity of the taste alterations likely to be encountered. Acknowledgments—The authors wish to thank Dr. Rona Delay for her generous help with the histological analysis of the taste epithelium and critical comments on the manuscript. They also want to thank Ben Eschle and Melissa Bainbridge for help with the behavioral studies, Jeremy Arenos for help with image capturing and processing, Brittany Carroll for help with the pilocarpine test, and Dr. Alan Howard for his help with SPSS. This work was supported in part by Vermont Genetics Network grant P20 RR16462 and by NSF grant IOS-0951016.

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(Accepted 6 July 2011) (Available online 18 July 2011)