British Journal of Pharmacology (2003) 139, 381-387© 2003 Nature Publishing Group All rights reserved 0007-1188/03 $25.00
`www.nature.com/bjp
`
`Effects of methyl /?-hydroxybenzoate (methyl paraben) on Ca2 +
`concentration and histamine release in rat peritoneal mast cells
`
`'Sanae Fukugasako, *,1Shinichi Ito & ’Yoshimi Ikemoto
`
`’Department of Dental Anesthesiology, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan
`
`1 Mechanisms of methyl /j-hydroxybenzoate (methyl paraben) action in allergic reactions were
`investigated by measuring the intracellular Ca2+ concentration ([Ca2+]i) and histamine release in rat
`peritoneal mast cells (RPMCs).
`In the presence or absence of extracellular Ca2+, methyl paraben (0.1 - 10mM) increased [Ca2+]j,
`2
`in a concentration-dependent manner. Under both the conditions, methyl paraben alone did not evoke
`histamine release.
`In RPMCs pretreated with a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate
`3
`(PMA) 3 and 10nM), methyl paraben (0.3- 3 mM) induced histamine release. However, a high
`concentration (10 mM) of the agent did not increase the histamine release.
`4 U73122 (0.1 and 0.5 ^M), an inhibitor of phospholipase C (PEC), significantly inhibited the methyl
`paraben-induced histamine release in PMA-pretreated RPMCs. U73343 (0.5 ^M), an inactive
`analogue of U73122, did not inhibit the histamine release caused by methyl paraben.
`In Ca2+-free solution, PEC inhibitors (U73122 0.1 and 0.5/iM, D609 l-10^M) inhibited the
`5
`methyl paraben-induced increase in [Ca2+]i, whereas U73343 (0.5 jzM) did not.
`6 Xestospongin C (2-20^M) and 2 aminoethoxydiphenyl borate (30 and 100/iM), blockers of the
`inositol 1,4,5-trisphosphate (IP3) receptor, inhibited the methyl paraben-induced increase in [Ca2+]; in
`Ca2+-free solution.
`In conclusion, methyl paraben causes an increase in [Ca2+]j, which may be due to release of Ca2 +
`7
`from storage sites by IP, via activation of PEC in RPMCs. In addition, methyl paraben possibly has
`some inhibitory effects on histamine release via unknown mechanisms.
`British Journal of Pharmacology (2003) 139, 381-387. doi: 10.1038/sj.bjp.0705248
`Methyl /i-hydroxybenzoate; mast cells; intracellular calcium concentration; histamine release; phospholipase C;
`protein kinase C; inositol 1,4,5-trisphosphate
`2APB, 2 aminoethoxydiphenyl borate; [Ca2+]i, intracellular Ca2+ concentration; DM SO, dime­
`thyl sulphoxide; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; PEC, phospholipase C; PM A, phorbol
`12-myristate 13-acetate; PSS, physiological salt solution; RPMCs, rat peritoneal mast cells
`
`Keywords:
`
`Abbreviations:
`
`Introduction
`
`There have been numerous reports on cases of anaphylactic
`reactions caused by various drugs (Fisher & More, 1981;
`Merles & Laxenaire, 2002). Anaphylactic shock is the type I
`allergy reaction mediated by IgE antibodies and mast cells.
`The symptom similar to anaphylactic shock is called an
`anaphylactoid reaction (Fisher & Pennington, 1982), where the
`mechanism involves activation of mast cells without IgE
`antibodies (Stellato & Marone, 1995). A preservative, methyl
`p-hydroxybenzoate (methyl paraben), may be responsible for
`some cases of anaphylactic shock and anaphylactoid reactions
`caused by various commercially available medicines (Nagel
`et al., 1977; Wildsmith et al., 1998). Methyl paraben is
`nonstimulating and nontoxic, and has a broad antibiotic
`spectrum. The compound is widely used as a preservative for
`foods, cosmetics and medicines. Those methyl paraben-
`containing products caused contact dermatitis and drug
`hypersensitivity (Larson, 1977; Mowad, 2000), but there has
`
`’Author for correspondence; E-mail: ito@dent.kyushu-u.ac.jp
`
`been no fundamental study on allergic reactions related to
`methyl paraben.
`In an immunological mechanism, degranulation of mast
`cells is triggered off by the aggregation of high-affinity
`receptor for the Fc region of IgE (FcsRI) caused by
`crosslinking of IgE by polyvalent antigens. However,
`specific IgE antibodies for methyl paraben have not been
`identified (Kokubu et al., 1989). Simple chemicals such as
`methyl paraben are incapable of producing sensitization
`and induction of immediate or delayed hypersensitivity
`without prior conjugation to carrier molecules, usually
`proteins. The bound methyl paraben is then considered
`a hapten, whereas its chemical properties are not clear
`(Soni et al., 2002).
`It was reported that methyl paraben activated the ryanodine
`receptor Ca2+ release channel in skeletal muscle terminal
`cisternae (Cavagna et al., 2000). On the other hand, Teraoka
`et al. (1997) reported that caffeine, an activator of the
`ryanodine receptor, did not increase the intracellular Ca2 +
`concentration ([Ca2+]i) in rat peritoneal mast cells, and
`Soboloff & Berger (2002) described that
`I
`I
`
`EXHIBIT
`,/^y
`
`Apotex (IPR2019-00400) Ex. 1028 p. 001
`
`

`

`382
`
`S. Fukugasako et al Methyl paraben on Ca2+ mobilization and secretion
`
`significantly increase [Ca2+]i in bone marrow-derived mast cells.
`However, the lack of stimulatory effects of caffeine and
`ryanodine on Ca2+ release does not accordingly indicate the
`absence of ryanodine receptor in many types of nonexcitable cells
`(Hosoi et al., 2001). The existence of ryanodine receptor is still
`under controversy, raising the question as to how methyl paraben
`affects the intracellular events during the allergic reactions.
`In the present study, in order to clarify the mechanism of
`allergic reactions caused by methyl paraben, we investigated
`the effects of the agent on the changes in [Ca2+]i and histamine
`release in RPMCs.
`
`Methods
`
`Mast cell isolation and purification
`
`Male Sprague - Dawley rats weighing 400 - 800 g were anaes­
`thetized with diethyl ether, and then killed by bleeding from
`the carotid arteries. Rat peritoneal mast cells were isolated and
`purified over Percoll density gradient as previously described
`(Chan et al., 2000). The peritoneal cavity was injected with the
`physiological salt solution (PSS) containing BSA
`(0.3 mg ml-1). After gentle massage of the rat abdominal
`region, mixed peritoneal cells (30-35 ml of fluid) were
`obtained by peritoneal lavage. The mixed peritoneal cells were
`then washed twice by centrifugation (1100 r.p.m., 5 min, 4°C)
`and were resuspended in 1 ml of PSS. The cell suspension was
`then mixed with 2 ml of 33% Percoll. BSA-supplemented PSS
`(1 ml) was then carefully layered over the Percoll-cell mixture.
`Purification was then performed by centrifugation (3000
`r.p.m., 20 min, 4°C), which allowed cell separation and
`gradient formation. Harvesting of the mast cells posed no
`problem since these cells gathered in a layer at the bottom of
`the tube, whereas other cells formed a rather compact layer on
`top of the gradient and could easily be removed by aspiration.
`The cell fraction was then washed twice in PSS by centrifuga­
`tion and finally resuspended to the desired cell density in PSS.
`
`Intracellular calcium measurements
`
`Intracellular Ca2+ images were obtained with the confocal
`laser scanning microscope (1X70, Olympus). Cells were
`incubated in PSS containing the acetoxymethyl ester of fluo-
`3 (fluo-3 AM 5 fiM) for 30 min at room temperature (22-
`25°C). Cells attached to a glass coverslip were mounted on the
`bottom of a chamber of 500 ;d capacity and placed in the
`microscope for fluorescence measurements. Cells were excited
`at 488 nm with an argon laser beam through the objective lens
`(UplanApo40X, Olympus). Fluo-3 fluorescent images (emis­
`sion 530 nm) were collected with the scan unit (FVX-SU,
`Olympus) every 0.42 s. To estimate [Ca2+]i, the mean intensity
`of cell area except nucleus was calculated with the analysing
`software (FLUOVIEW FV500, Olympus). The data were
`expressed as ratios of fluorescence intensity changes (F)
`relative to control values before stimulation (Fo), namely
`(F-Fo) Fo1.
`
`Histamine measurements
`
`Drugs were applied to cell suspensions (106 cells mF1), making
`a total of 1 ml solution. The histamine-releasing reactions were
`
`British Journal of Pharmacology vol 139 (2)
`
`terminated by placing the test tube in ice-cold water for 10 min.
`Cell suspensions were then centrifuged and divided into
`fractions of supernatant (0.5 ml) and supernatant plus pellets
`(0.5 ml), both of which were acidified with perchloric acid
`(50/zl) to abolish histamine breakdown. After both fractions
`were centrifuged at 1100 r.p.m. for 10 min at 4°C to remove
`proteins, secreted histamine was determined by the fluoro­
`metric method. Histamine release was expressed as a
`percentage of the total cell contents. All samples were stored
`at -25°C until the histamine level was measured using the
`high-performance liquid chromatography (HPLC) postlabel
`system as previously described (Yamatodani et al., 1985;
`Horinouchi et al., 1993). The system was composed of an
`intelligent pump (Hitachi, L-6200), a reaction pump (Hitachi,
`655-A-13), a fluorescence spectrophotometer (Hitachi, F-
`1150), an autosampler (Hitachi, AS-4000), a Chromato­
`integrator (Hitachi, D-2500) and a 6 0, 15 cm column
`(Catecholepak, Toyosoda, Tokyo, Japan) warmed at 50°C
`by a column oven (Hitachi, L-5020). Each 100/zl of super­
`natant was injected into the HPLC for each sample. The
`excitation wavelength used was 340 nm and the emission was
`450 nm.
`
`Chemicals
`
`The drugs used were BSA, EGTA, methyl paraben, HEPES,
`phorbol 12-myristate 13-acetate (PMA), compound 48/80,
`U73343 and U73122 (Sigma Chemicals, St Louis, U.S.A.),
`dimethyl sulphoxide (DMSO) (Merck, Darmstadt, Germany),
`A23187, 2APB, xestospongin C and D609 (Calbiochem, San
`Diego, CA, U.S.A.), Fluo-3 AM (Dojindo Laboratories,
`Kumamoto, Japan) and Percoll (Pharmacia Fine Chemicals,
`Uppsala, Sweden).
`
`Solutions
`
`The PSS contained (in mM): NaCl 138.6, KC1 5.0, MgCI2 1.5,
`CaCl2 2.0, HEPES 10, glucose 5.6, KH2PO4 1.0, pH 7.2. Ca2+-
`free solution was made by substituting an equimolar concen­
`tration of MgCl2 instead of CaCl2 and adding 0.5 mM EGTA.
`
`Statistics
`
`Data were expressed as mean + s.e.m. and statistical signifi­
`cance was determined using the paired or unpaired Student’s
`/-test. Probabilities less than 5% (P<0.05) were considered
`significant.
`
`Results
`Methyl paraben-induced increase in [Ca2+/,• in the
`presence or absence of external Ca2+
`
`Figure la and b shows representative traces of the effects of
`methyl paraben (3mM) on [Ca2+]j in rat peritoneal mast cells
`(RPMCs). Methyl paraben was applied for 75 s. The agent
`produced a transient increase in [Ca21 ], both in Ca2+-
`containing solution (PSS, la) and in Ca2+-free solution (lb).
`Figure 1c shows the average of the peak values of the changes
`in [Ca2+]i induced by methyl paraben (O.l-lOmM). The
`[Ca2+]i was increased concentration-dependently in the pre­
`
`Apotex (IPR2019-00400) Ex. 1028 p. 002
`
`

`

`S. Fukugasako et al Methyl paraben on Ca2+ mobilization and secretion
`
`383
`
`Figure 1 Effects of methyl paraben on [Ca2+]i in RPMCs. The fluo-3 AM-loaded RPMCs were stimulated with methyl paraben for
`75 s at room temperature (22-25°C). (a) Methyl paraben (3mM) increased the [Ca2+]i in an RPMC incubated in PSS containing
`2mM Ca2+. (b) Methyl paraben was applied in Ca2 ' -free solution containing 0.5 mM EGTA after a 5 min removal of Ca2+. Also in
`Ca2+-free condition, methyl paraben increased the [Ca2 ' ], in an RPMC. (c) The Concentration - response relation of methyl
`paraben-induced [Ca 2+]j increase in Ca2+-containing (open circles) and Ca2+-free (filled circles) mediums. The ordinate shows the
`net maximum [Ca2+]; of the response with baseline subtracted. The data points indicate mean + s.e.m. of 20 experiments. The
`asterisk shows a significant difference in Ca2+-containing and Ca2+-free solutions.
`
`sence of extracellular Ca2 +. A similar result was obtained also
`in the Ca2+-free solution. At a high concentration (10mM),
`however, the increase was significantly greater in the Ca2+-
`containing solution.
`
`Methyl paraben did not evoke histamine release from
`RPMCs
`
`The amount of histamine release was measured as an index of
`the degranulation of RPMCs and illustrated in Figure 2. The
`basal spontaneous release was around 10% of the total contents
`in RPMCs (control). Compound 48/80 (0.3 ^g ml-1) potentiated
`the histamine release both in PSS and in Ca2+-free solution,
`increasing it up to 63.9 + 5.2% (n = 4) and 34.6+1.9% (n = 4),
`respectively. A combination of a Ca2+ ionophore (A23187
`0.1 /zM) and the phorbol ester (PMA lOnM) caused the release
`of histamine in PSS (40.9+1.2%, n = 5). In contrast, methyl
`paraben did not increase histamine release in PSS (4.32 + 0.58%,
`n = 4, by 3 mM and 9.84+ 1.70%, n = 4, by 10 mM). It was also
`the case in Ca2+-free solution (6.49+0.91%, w = 4, by 3mM and
`8.92+1.18%, n = 4, by 10mM).
`
`Methyl paraben-evoked histamine release from PMA-
`pretreated RPMCs
`
`Incapability of methyl paraben to release histamine in the
`present study may be due to insufficient activation of protein
`kinase C (PKC), since it has been reported that both increase
`in [Ca2+]i and activation of PKC are needed for the release
`(Beaven & Cunha-Melo, 1988). We, therefore, pretreated
`RPMCs with PM A for 5 min to activate PKC. The sponta­
`
`neous histamine release was normalized as a relative histamine
`release of 1.0. Application of methyl paraben alone at 0.3-
`lOmM did not induce histamine release (open circles). In the
`absence of methyl paraben (control), PMA at 3nM (closed
`circles) and 10 nM (closed squares) increased the release to
`1.17 + 0.30 (n = 4) and 2.05+0.54 (w = 4) fold, respectively.
`Pretreatment with PMA for 5 min greatly enhanced the
`histamine release evoked by methyl paraben (0.3-10mM).
`The peak increase was 2.23 + 0.11 times the control increase in
`the presence of 3 nM PMA (n = 4, 1 mM methyl paraben) and
`1.53 + 0.53 times the control increase in the presence of lOnM
`PMA (n = 4, 3mM methyl paraben). A high concentration
`(10 mM) of methyl paraben, however, did not augment or even
`suppressed the histamine release from the PMA-pretreated
`RPMCs. The 10 nM PMA-induced increase in histamine
`release was suppressed to 0.79+0.18 (n = 4) of the control
`increase (Figure 3).
`
`Effects of U73122 and U73343 on histamine release
`induced by combination of PMA and methyl paraben
`
`To assess the involvement of phospholipase C (PLC) in the
`histamine release reaction by methyl paraben in the PMA-
`pretreated RPMCs, effects of a PLC inhibitor U73122 and its
`inactive analogue U73343 were examined. The spontaneous
`histamine release in PSS containing 2mM Ca2+ was normal­
`ized as a relative histamine release of 1.0. A volume of 10 nM of
`PMA increased the release to 1.80 + 0.17 fold (n=12), and
`U73122 (0.5 pM) did not significantly inhibit the increase
`(n=12). The histamine release caused by methyl paraben
`(3mM) applied 5 min after application of PMA (10 nM) was
`
`British Journal of Pharmacology vol 139 (2)
`
`Apotex (IPR2019-00400) Ex. 1028 p. 003
`
`

`

`384
`
`S. Fukugasako et al Methyl paraben on Ca2+ mobilization and secretion
`
`control
`
`Figure 2 Effects of methyl paraben (3 and 10 him) and various
`stimuli on histamine release from RPMCs. Cells were preincubated
`for 5 min at 37°C and subsequently incubated with methyl paraben
`(3 and 10mM), compound 48/80 (0.3/ig ml"1) or a combination of
`A23187 (0.1 /im) and PMA (10nM) for 30min in the external
`solution with (open columns) or without (closed columns) Ca2+. The
`released histamine in the 30 min period was calculated as a
`percentage of the total histamine content of the cells. Results are
`expressed as mean + s.e.m. of eight to 10 experiments.
`
`-O- PMA(-)
`
`0 J----- 1——’----- '—' ■ ■ ■ ' i--------------- ■-------- •---- ’—...................
`1
`control 0.3
`3
`10
`methyl paraben (mM)
`Figure 3 Effects of combined application of methyl paraben and
`PMA on histamine release in PSS. Cells were preincubated for 5 min
`at 37°C in the external solution with Ca2+ and subsequently
`incubated without (open circles) or with PMA (3 nM, closed circles
`or 10 nM, closed squares) for 5 min. The cells were then stimulated
`with methyl paraben (3 mM) for 30 min in the continuous presence
`or absence of PMA. The spontaneous histamine release was used for
`normalization as a relative histamine release of 1.0. The symbols
`refer to the mean of four to 12 experiments and the error bars
`represent s.e.m.
`
`3.86±0.28 times the control (n = 5). U73122 significantly
`reduced this augmented histamine release by the combination
`of PMA and methyl paraben. The normalized inhibitory value
`by U73122 on the combination of PMA and methyl paraben-
`induced augmentation was 0.68 + 0.04 (w = 5) and 0.10 + 0.02
`(n = 5) at 0.1 and 0.5/zM, respectively. In contrast,
`U73343 (0.5 /zM) did not inhibit the histamine release caused
`by the combined application of PMA and methyl paraben
`(Figure 4).
`
`British Journal of Pharmacology vol 139 (2)
`
`Figure 4 Effects of U73122 and U73343 on histamine release
`induced by a combination of PMA and methyl paraben. After
`preincubation for 5 min at 37°C in PSS, cells were pretreated with
`PMA (10 nM) or a combination of PMA and U73122 (0.1 and
`0.5 /iM) or U73343 (0.5 /tM) for 5 min. The cells were then stimulated
`with methyl paraben (closed columns) or not (hatched columns) and
`incubated for 30 min in PSS with continued treatment of PMA in the
`continuous presence or absence of U73122 or U73343. The
`spontaneous histamine release was used for normalization as a
`relative histamine release of 1.0 (open column). Results are
`expressed as mean + s.e.m. of five to 12 experiments. Asterisks
`indicate a significant difference from the combined application of
`PMA and methyl paraben in the absence of U73122 or U73343 with
`P<0.05.
`
`Effects of U73122, U73343 and D609 on increase in
`[ Ca2+]i induced by methyl paraben
`
`We investigated the effects of U73122, U73343 and another
`PLC inhibitor D609 to confirm that the increase in [Ca2+]i
`caused by methyl paraben was induced through activation of
`PLC in RPMCs. The change in [Ca2+]i caused by 3mM of
`methyl paraben in the Ca2 1-free solution containing 0.5 mM
`EGTA was used as a control (Figure 5a). Pretreatment with
`U73122 (0.5/zm) for 5 min markedly inhibited the [Ca2+]i
`increase, while U73343 (0.5/tM) did not (Figure 5b). D609 at
`10 [M completely suppressed the increase (Figure 5c). Figure 5d
`illustrates the effects of U73312 (0.1 and 0.5/zM), U73343
`(0.5/zM) and D609 (1 - 10 /zM) on the peak increase in [Ca2+]j
`caused by 3mM of methyl paraben. U73122 (0.1 and 0.5/zM)
`and D609 (1 -10/zM) inhibited the increases in [Ca2+]i in a
`concentration-dependent manner, whereas the inactive analo­
`gue (U73343 0.5/zM) did not.
`
`Effects of xestospongin C and 2APB on increase in
`[Ca2+f induced by methyl paraben
`
`PLC increases the cytosolic levels of inositol 1,4,5-trispho-
`sphate (IP3) and DAG in mast cells (White et al., 1985). IP3
`stimulates release of Ca2+ from internal stores (Meyer et al.,
`1988; Berridge, 1993) and DAG is known to activate PKC
`(Nishizuka, 1984). We investigated the effects of xestospongin
`C and 2 aminoethoxydiphenyl borate (2APB), inhibitors of
`IP3-induced Ca2+ release, on the methyl paraben-induced
`increase in [Ca2+]i in RPMCs. Figure 6a depicts a control
`increase in the [Ca2+]i induced by methyl paraben (3mM) in
`the Ca2 + -free solution containing 0.5 mM EGTA. Pretreat-
`
`Apotex (IPR2019-00400) Ex. 1028 p. 004
`
`

`

`S. Fukugasako et al Methyl paraben on Ca2+ mobilization and secretion
`
`385
`
`10pMD609
`
`3 mM methyl paraben
`^~90
`30
`60
`time (s)
`
`0
`
`Figure 5 Effects of U73122, U73343 and D609 on the increase in [Ca2+]j induced by methyl paraben in Ca2+-free solution
`containing 0.5 mM EGTA. Methyl paraben (3 mM) was applied for 60 - 75 s following a 5 min incubation in the Ca2 + -free solution,
`(a) Methyl paraben increased the [Ca2 * ], in a fluo-3 AM-loaded RPMC. (b) Pretreatment good for 5 min with U73122 (0.5/iM)
`markedly suppressed the increase, while U73343 (0.5/iM) had no effects, (c) Pretreatment for 5 min with D609 (10/<M) completely
`blocked the effect of methyl paraben, (d) Each column indicates the average of the peak increase above the base line (n = 8- 10).
`U73122 and D609 suppressed the [Ca2+]i increase dose-dependently. The reduction by U73343 was not significant. Bars indicate
`s.e.m. Asterisks indicate significant difference from the control with Pc0.05.
`
`Figure 6 Effects of xestospongin C and 2APB on the increase in [Ca2+]i induced by methyl paraben in Ca2+-free solution
`containing 0.5 mM EGTA. Methyl paraben (3 mM) was applied for 70 - 75 s in Ca2+-free solution after a 5 min removal of Ca2+. (a)
`Methyl paraben increased the [Ca2+]i in a fluo-3 AM-loaded RPMC. (b) Pretreatment for 5 min with xestospongin C (6 or 20/iM)
`markedly suppressed the [Ca2+]i increase, (c) Pretreatment for 5 min with 2APB (100 /iM) greatly decreased the [Ca2+]j increase, (d)
`Each column indicates the average of the peak increase above the base line (n = 7 - 10). Xestospongin C and 2APB dose-dependently
`suppressed the [Ca2+]i increase produced by methyl paraben. Bars indicate s.e.m. Asterisks show significant suppression from the
`control with P<0.05.
`
`ment with xestospongin C (6 and 20;tM; Figure 6b) or 2APB
`(100 /zM; Figure 6c) markedly inhibited the increase in [Ca2+]j.
`Effects of the IP3 inhibitors on the peak increase in [Ca2+]i are
`
`shown in Figure 6d. Xestospongin C (2-20^M) and 2APB (30
`and 100/zM) suppressed the increases in [Ca2+]i in a
`concentration-dependent manner.
`
`British Journal of Pharmacology vol 139 (2)
`
`Apotex (IPR2019-00400) Ex. 1028 p. 005
`
`

`

`386
`Discussion
`
`In this study, we investigated the effects of methyl paraben on
`the changes in [Ca2+]i and histamine release in RPMCs.
`Methyl paraben at 1-lOmM dose-dependently increased
`[Ca2+]j in both Ca2+-containing and Ca2+-free solutions.
`The peak increase in [Ca2+]j induced by 0.3- 3 mM of methyl
`paraben was not significantly different between the presence
`and absence of the external Ca2+. This finding suggests that
`the transient increase in [Ca2+]i by the agent was largely due to
`release of Ca2+ from intracellular storage sites. At a high
`concentration (10mM), however, the peak increase was
`significantly greater in the Ca2+-containing solution. High
`concentrations of methyl paraben may induce Ca2+ influx
`from the extracellular medium (Figure 1).
`Many reports have demonstrated that Ca2+ chelators such
`as EGTA increase [Ca2' ], in mast cells, although the reason is
`not known (Teraoka et al., 1997). In the present experiments
`too, EGTA caused an increase in [Ca2+]i in some of the mast
`cells. However, this reaction was observed only within 5 min of
`the exposure to EGTA (data not shown). Hence, measure­
`ments were made 5 min after replacement of PSS with Ca2+-
`free solution, and cells showing no change in [Ca2+]i were
`selected for the following experiments. Actually, changes in
`[Ca2+]i were negligible probably due to the low concentration
`of EGTA (0.5 mM) in this study.
`Application of the stimulants known to release histamine
`from mast cells, such as compound 48/80, known as an
`activator of PEC (Cockcroft & Gomperts, 1979), and the
`combination of A23187 and PM A (Chew et al., 2002), released
`histamine in our experimental system, but methyl paraben at
`0.3- 10 mM did not induce histamine release (Figures 2 and 3).
`Dvorak et al. (1996) demonstrated that the interaction between
`inositol phospholipids and PKG regulated degranulation of
`mast cells. In addition, Akita et al. (1990) reported that
`Ca2+ played an important role in the binding of PKC to
`the cell membrane. Furthermore, it was suggested that
`PKC was activated by increased [Ca2+]i and phospholipids,
`and that the activation was further enhanced by DAG
`(White et al., 1985). Thus, the increase in [Ca2+]j and
`the activation of PKC, which may lead to degranulation,
`are required to release histamine (Heiman & Crews, 1985a, b).
`Application of methyl paraben alone did not induce histamine
`release in the present experiments, but after activation of
`PKC by PMA, the agent evoked histamine release, which
`might be mediated by the activated PKC and the
`Ca2+ mobilized by methyl paraben. The increase in [Ca2+]i
`induced by methyl paraben was dose-dependent (Figure 1),
`while the release of histamine by the combined application of
`PMA and methyl paraben was not dose-dependent but
`inhibited at the higher concentration (10mM) (Figure 3).
`These results suggest that methyl paraben not only mobilizes
`Ca2+ but also inhibits the histamine release via some unknown
`mechanisms.
`The PEC inhibitors, U73122 (Bleasdale et al., 1990) and
`D609 (Ito et al., 2002), inhibited the increase in [Ca2+]i caused
`by methyl paraben. U73122 also inhibited the histamine
`release induced by the combined application of PMA and
`methyl paraben. In contrast, U73343, an inactive analogue of
`U73122 (Pocock & Bates, 2001), did not show any effects on
`these reactions. In addition, U73122 did not significantly
`suppress the histamine release caused by PMA (Figures 4 and
`
`British Journal of Pharmacology vol 139 (2)
`
`S. Fukugasako et al Methyl paraben on Ca2+ mobilization and secretion
`
`5). Thus the PEC inhibitors suppressed the action of methyl
`paraben, indicating that methyl paraben causes the increase in
`[Ca2+]i via activation of PEC. When PEC is activated in mast
`cells, phosphatidylinositol 4,5-bisphosphate (PIP2) is hydro­
`lysed to produce IP3 and DAG. IP3 binds to its receptors on
`the intracellular Ca2+ storage site to release Ca2+ (Meyer et al.,
`1988; Berridge, 1993), while DAG activates PKC (White &
`Metzger, 1988). 1P3 receptor blockers, xestospongin C (Gafni
`et al., 1997) and 2APB (Maruyama et al., 1997), inhibited the
`increase in [Ca2+]j induced by methyl paraben (Figure 6),
`suggesting that methyl paraben produced IP3 through PEC
`activation, which resulted in a release of Ca2+ from the
`intracellular storage sites in RPMCs. 4-Chloro-/n-cresol (0.5
`and 1 mM), a potent activator of the ryanodine receptor
`(Zorzato et al., 1993), induced the transient increase in [Ca2+];;
`however, additional application of any stimulants such as
`compound 48/80, methyl paraben or A23187 had no effect on
`[Ca2+]j in RPMCs (data not shown). These results suggest the
`colocalization of ryanodine receptor and IP3 receptor in Ca2+
`store. Although the effect of methyl paraben on the ryanodine
`receptor in RPMCs is still unclear, PEC inhibitors and IP3
`receptor blockers almost completely suppressed the action of
`methyl paraben (Figures 4-6), indicating that IP3 is a key
`messenger on Ca2+ release and subsequent histamine release
`induced by methyl paraben in RPMCs. DAG produced by
`PEC activation is expected to activate PKC and to evoke
`degranulation by acting together with mobilized Ca2+.
`However, no histamine release was observed by methyl
`paraben alone in the present study (Figures 2 and 3). In
`addition, methyl paraben released histamine from the mast
`cells in which PKC had been activated by PMA (Figure 3).
`These arguments suggest that although methyl paraben
`activates PEC, the PKC activity is not enough to release
`histamine in RPMCs.
`Methyl paraben is contained in various pharmaceutical
`agents at certain concentrations: 0.06-0.25% in injections;
`0.015-0.2% in syrups; 0.015-0.05% in instillations; and
`0.02-0.3% in external medicines (Anonymous, 1998). In
`addition, methyl paraben is added to cosmetics at 0.32% at
`maximum (Rastogi et al., 1995), and to food at 0.03-0.07% in
`combination with propyl paraben (Krebs-Smith et al., 1997).
`In this study, methyl paraben showed a significant effect on the
`[Ca2+]j increase and histamine release at the concentrations of
`1 -3mM, which correspond to approximately 0.015-0.046%.
`Therefore, some agents containing methyl paraben may induce
`an increase in [Ca2+]j when directly acting on mast cells.
`The molecular weight of methyl paraben is as small as
`152.14, and hence it has been thought that the agent by itself
`does not have antigenicity and may become antigenic when
`bound to a certain kind of proteins (Pressman et al., 1968). In
`the present experiments, methyl paraben might act on mast
`cells with no relation to FcsRI on account of the absence of
`specific IgE and proteins to bind. In vivo, however, the
`situation would be different from in vitro, and it is likely that
`methyl paraben specific IgE may mediate the activation of
`Ca2+ release and subsequent histamine release in mast cells. In
`addition, it is not clear whether methyl paraben bound to
`proteins has an inhibitory action on histamine release; there­
`fore, the usual view that methyl paraben induces allergic
`reactions through an immunological mechanism is not
`excluded. On the other hand, the present results propose the
`possibility that methyl paraben may cause anaphylactoid
`
`Apotex (IPR2019-00400) Ex. 1028 p. 006
`
`

`

`reactions through a nonimmunological mechanism when PKC
`is simultaneously activated to some extent in the mast cells.
`
`We thank Prof. Y. Ito (Department of Pharmacology, Graduate
`School of Medical Sciences, Kyushu University) for his advice.
`
`S. Fukugasako et al Methyl paraben on Ca2+ mobilization and secretion
`
`387
`
`References
`
`AKITA, M„ SASAKI, S„ MATSUYAMA, S. & MIZUSHIMA, S. (1990).
`SecA interacts with secretory proteins by recognizing the positive
`charge at the amino terminus of the signal peptide in Escherichia
`coli. J. Biol. Chem., 265, 8164- 8169.
`ANONYMOUS (1998). Preservation, sterilization and stability testing
`of ophthalmic preparations. Ini. J. Pharm. Compounding, 2, 192 —
`195.
`BEAVEN, M.A. & CUNHA-MELO, J.R. (1988). Membrane phosphoi­
`nositide-activated signals in mast cells and basophils. Prog. Allergy,
`42, 123 - 184.
`BERRIDGE, M.J. (1993). Inositol trisphosphate and calcium signalling.
`Nature, 361, 315-325.
`BLEASDALE, J.E., THAKUR, N.R., GREMBAN, R.S., BUNDY, G.L.,
`FITZPATRICK, F.A., SMITH, R.J. & BUNTING, S. (1990). Selective
`inhibition of receptor-coupled phospholipase C-dependent pro­
`cesses in human platelets and polymorphonuclear neutrophils. J.
`Pharmacol. Exp. Ther., 255, 756-768.
`CAVAGNA, D., ZORZATO, F., BABINI, E., PRESTIPINO, G. &
`TREVES, S. (2000). Methyl p-hydroxybenzoate (E-218) a preserva­
`tive for drugs and food is an activator of the ryanodine receptor
`Ca2+ release channel. Br. J. Pharmacol., 131, 335-341.
`CHAN, C.L., JONES, R.L. & LAU, H.Y. (2000). Characterization of
`prostanoid receptors mediating inhibition of histamine release from
`anti-IgE-activated rat peritoneal mast cells. Br. J. Pharmacol., 129,
`589 - 597.
`CHEW, C.S., CHEN, X., PARENTE Jr J.A., TARRER, S., OKAMOTO, C.
`& QIN, H.Y. (2002). Lasp-1 binds to non-muscle F-actin in vitro and
`is localized within multiple sites of dynamic actin assembly in vivo.
`J. Cell. Sci., 115, 4787-4799.
`COCKCROFT, S. & GOMPERTS, B.D. (1979). Evidence for a role of
`phosphatidylinositol turnover in stimulus-secretion coupling. Bio-
`chem. J., 178, 681 -687.
`DVORAK, A.M., ACKERMAN, S.J., LETOURNEAU, L„ MORGAN,
`E.S., LICHTENSTEIN, L.M. & MACGLASHAN Jr. D.W. (1996).
`Vesicular transport of Charcot - Leyden crystal protein in tumor­
`promoting phorbol diester-stimulated human basophils. Lab.
`Invest., 74, 967 - 974.
`FISHER, M.M. & MORE, D.G. (1981). The epidemiology and clinical
`features of anaphylactic reactions in anaesthesia. Anaesth. Intens.
`Care, 9, 226 - 234.
`FISHER, M.M. & PENNINGTON, J.C. (1982). Allergy to local
`anaesthesia. Br. J. Anaesth., 54, 893 - 894.
`GAFNI, J., MUNSCH, J.A., LAM, T.H., CATLIN, M.C., COSTA, L.G.,
`MOLINSKI, T.F. & PESSACH, LN. (1997). Xestospongins: potent
`membrane permeable blockers of the inositol 1,4,5-trisphosphate
`receptor. Neuron, 19, 723 - 733.
`HEIMAN, A.S. & CREWS, F.T. (1985a). Characterization of the effects
`of phorbol esters on rat mast cell secretion. J. Immunol., 134, 548 -
`555.
`HEIMAN, A.S. & CREWS, F.T. (1985b). Hydrocortisone inhibits
`phorbol ester stimulated release of histamine and arachidonic acid
`from rat mast cells. Biochem. Biophys. Res. Common., 130, 640-
`645.
`HOR1NOUCHI, Y., ABE, K., KUBO, K. & OKA, M. (1993). Mechanisms
`of vancomycin-induced histamine release from rat peritoneal mast
`cells. Agents Actions, 40, 28 - 36.
`HOSOI, E., NISHIZAKI, C., GALLAGHER, K.L., WYRE, H.W.,
`MATSUO, Y. & SEI, Y. (2001). Expression of the ryanodine
`receptor isoforms in immune cells. J. Immunol., 167, 4887 - 4894.
`ITO, J., NAGAYASU, Y., UENO, S. & YOKOYAMA, S. (2002).
`Apolipoprotein-mediated cellular lipid release requires replenish­
`ment of sphingomyelin in a phosphatidylcholine-specific
`phospholipase C-dependent manner. J. Biol. Chem., 277,
`44709-4