International Journal of Zoological Investigations Vol. 3, No. 1, 72-81 (2017)______________________________________________________________________________________

International Journal of Zoological
Investigations
Contents available at Journals Home Page: www.ijzi.net
ISSN: 2454-3055

Benz[a]anthracene Decreases Plasma Calcium Levels Resulting from
Influence of Scale Osteoclastic and Osteoblastic Activities in Goldfish
Nobuo Suzuki1, Jun Nakano 2, Kimi Kawabe2, Akira Toriba2, Kazuichi Hayakawa3, Ning Tang3,
Toshio Sekiguchi1, Yoshiaki Tabuchi4, Mika Ikegame5,6, Nobuaki Shimizu1, Hiroyuki Mishima7,
Atsuhiko Hattori8, Ajai K. Srivastav9 and Yoichiro Kitani1*
1Noto

Marine Laboratory, Institute of Nature and Environmental Technology, Division of Marine Environmental Studies,
Kanazawa University, Noto-cho, Ishikawa 927-0553, Japan
2Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma, Ishikawa 920 -1192, Japan
3Institute of Nature and Environmental Technology, Division of Atmosphere Environmental Studies, Kanazawa University,
Kakuma, Ishikawa 920-1192, Japan
4Division of Molecular Genetics Research, Life Science Research Center, University of Toyama, Sugitan i, Toyama 930-0194,
Japan
5Department of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama, Okayama 700-8525, Japan
6ARCOCS, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Okayama 7008525, Japan
7Department of Dental Engineering, Tsurumi University School of Dental Medicine, Yokohama 230 ‑ 8501, Japan
8Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827,
Japan
9Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273-009, India*Corresponding Author
Received: 8 th June 2017
Accepted: 26th June 2017__________________________________________________________________________________________________________
Abstract: In the present study, the effects of polycyclic aromatic hydrocarbons (PAHs) on plasma calcium, osteoblasts andosteoclasts were investigated. Goldfish were intraperitoneally injected with benz[a]anthracene (BaA) (5µg/g body weight)(around 10 -5 M). BaA induced hypocalcemia at 24 and 48 h and thereafter the level recovered to control levels at 72 h. Goldfish
scales possess both osteoclasts (bone resorption cells) and osteoblasts (bone forming cells). The marker e nzyme activity(tartrate-resistant acid phosphatase: TRAP) of osteoclasts in goldfish scales decreased at 12 and 24 h after BaA injection. In BaA
injected goldfish scales, the marker enzyme (alkaline phosphatase, ALP) gradually decreased at 48 and 72 h. In addition,
4-hydroxybenz[a]anthracene (4-OHBaA) that is one of the metabolites of BaA by conversion enzyme (P4501A1) was detectedin the bile of goldfish at 12, 24, 48, and 72 h after administration of BaA to goldfish. We have recently found the toxicity ofmonohydroxylated polycyclic aromatic hydrocarbons (OHPAHs), metabolites of PAHs, in bone metabolism. We reported that 4OHBaA suppressed both TRAP and ALP activities in the cultured scales of goldfish. Therefore, it was prob able that 4-OHBaA
metabolized from BaA has toxicity for osteoclasts and osteoblasts in goldfish. These phenomena are a cause of the disruption of
the bone metabolism and the induction of spinal deformities.
Keywords: Benz[a]anthracene, 4-Hydroxybenz[a]anthracene, Teleost Scale, Osteoblasts, Osteoclasts, Calcium. Goldfish_________________________________________________________________________________________________________

Introduction
Polycyclic aromatic hydrocarbons (PAHs) areubiquitous environmental pollutants derived
from petroleum and produced by combustionof fossil fuel, wood and other organic
materials (Lima et al., 2003), as well as incigarette smoke (Lee et al., 2002). It has beenreported that PAHs affects bone metabolism
inmammals.
For
example,PAHs(benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene) present in cigarette smoke
induced bone loss in an ovariectomized rat(Lee et al., 2002). In addition, PAHs inhibited
osteoclastogenesis in rabbit osteoclasts andthe RAW264.7 cells (mouse monocyte
macrophage cell line) (Voronov et al., 2005).
In the aquatic environment as well as
atmospheric environment, storm waterrunoff and atmospheric deposition are one ofsources for aquatic PAHs contamination(Lima et al., 2003; Li and Daler, 2004). In fact,spinal bone deformity was reported in pacificherring and pink salmon by PAHs (Barron etal., 2004; Billiard et al., 2006). Much attention
should be given to bone metabolism in fish.
It is known that the osteoclasts andosteoblasts in teleost scale are similarmorphological bone-like features which isfound in avian and mammalian membranebone (Yamada, 1961; Bereiter-Hahn and
Zylberberg, 1993; Suzuki et al., 2000, 2007,2008). The scales of some teleosts contain asmuch as 20% of the total body calcium andare thus a better potential internal calcium
reservoir rather than vertebral bone during
periods of increased calcium demand, such as
sexual maturation and starvation (Yamada,
1961; Berg, 1968; Mugiya and Watabe, 1977;
Bereiter-Hahn and Zylberberg, 1993). A co-

relation between mercury levels in the scales
and in the muscles was reported inlargemouth bass (Lake et al., 2006) althoughmercury did not accumulate in the vertebral
bone of fish (Camusso et al., 1995). Thisstrongly indicates that the scale is a more
active organ in bone metabolism thanvertebral bone. Considering these results, wedeveloped an original in vitro assay using
goldfish scales (Suzuki et al., 2000, 2007;
Suzuki and Hattori, 2002). This system cansimultaneously detect the activities of bothscale osteoclasts and osteoblasts withtartrate-resistant acid phosphatase (TRAP)
and alkaline phosphatase (ALP) as markers,
as shown by the fact that, in mammals, theeffects of hormones and some bioactive
substances on osteoclasts and osteoblasts
have been investigated using TRAP and ALP
as respective markers (Vaes, 1988; Dimai etal., 1998; Suda et al., 1999). We detected therespective enzyme activity from one scale bytransferring each scale into a 96-wellmicroplate and directly incubating it with thesubstrate in each well.
In the present study, we examined plasma
calcium levels and both scale osteoclastic andosteoblastic activities after intraperitoneal
injection of benz[a]anthracene (BaA)( 5µg/g
body weight) (around 10 -5 M) to goldfish.
Furthermore, the concentration of 4hydroxybenz[a]anthracene (4-OHBaA) that is
one of the metabolites of BaA by conversion
enzyme (P4501A1) was measured in the bileof BaA-injected goldfish because we
previously reported that 4-OHBaA inhibited
osteoclastic and osteoblastic activities in thecultured scales of goldfish (fresh water73

teleost) and wrasse (marine teleost)(Suzuki
et al., 2009a).
The present study is the first to
demonstrate that BaA decreases plasma
calcium levels in goldfish resulting frominfluence of osteoclasts and osteoblasts
activities in the scales of goldfish.
Materials and Methods
Animals:
Suzuki et al. (2000) reported that thesensitivity for calcemic hormones was higher
in mature female than in mature maleteleosts. Therefore, we have used female
goldfish (Carassius auratus) in the present
study which were purchased fromcommercial source (Higashikawa Fish Farm,
Yamatokoriyama, Japan). Fish were fed acommercial pellet diet every morning andwere kept in tap water at 26 C before thestart of experiment.
All experimental procedures wereconducted in accordance with the Guide for
the Care and Use of Laboratory Animals of
Kanazawa University.
Effects of BaA on plasma calcium levels in the
goldfish:In the experimental group, goldfish (n = 25)
were anesthetized with 0.03% ethyl 3aminobenzoate, methanesulfonic acid salt
(Sigma-Aldrich, Inc., MO, USA) and then BaA(Nakarai Chemicals Ltd., Kyoto, Japan) (5µg/g
body weight) (around 10 -5 M) was injected
intraperitoneally. BaA was firstly dissolved inethanol and then 0.9% NaCl was added tomake the desired stock solution. The goldfish
in the control group (n = 25) were injected
intraperitoneally with saline (0.9% NaClcontaining 0.1% ethanol) in the same manneras experimental goldfish. These goldfish were

kept in the aquarium for 72 h (3 days). This
experimental period was adopted because
hormonal and toxicological effects were
influenced in goldfish during 3 days (Suzuki
et al., 2004a, 2011; Omori et al., 2012;
Yachiguchi et al., 2014). During theexperimental periods, these goldfish were
fasted to exclude intestinal calcium uptake
from diets. After 12, 24, 48, and 72 h of theinjection, blood samples were collected fromthe caudal vessel of anesthetized goldfish byusing heparinized syringes from both control
and experimental groups at each intervalfrom each group (n = 5). The collected blood
was put into a 1.5 ml tube and centrifuged at
15,000 rpm for 3 min. Then, the separated
plasma was immediately frozen and kept at –80 C until use. The plasma total calcium
levels (mg/100 ml) were determined using
an assay kit (Calcium E, Wako Pure Chemical
Industries).
Effects of BaA on osteoclastic and osteoblastic
activities in the scales of goldfish:
Prior to BaA or saline (0.9% NaCl containing
0.1% ethanol) injection, the scales (TRAP for
8 scales; ALP for 8 scales) were removed
under anesthesia with 0.03% ethyl 3aminobenzoate, methanesulfonic acid salt
(Sigma-Aldrich). At 12, 24, 48, and 72 h after
BaA or saline (0.9% NaCl containing 0.1%
ethanol) injection, scales (TRAP for 8 scales;
ALP for 8 scales) were extracted fromanesthetized goldfish to examine theinfluences of BaA on the osteoclasts andosteoblasts with TRAP and ALP as respective
marker.
For measuring TRAP and ALP activities,
an aliquot of 100 μl of an acid buffer (0.1 M
sodium acetate, including 20 mM tartrate, pH
5.3) or an alkaline buffer (100 mM Tris-HCl,74

pH 9.5; 1 mM MgCl2) was added to each wellin a 96-well microplate. Then, each scale wasput into its separate well. This microplate
was immediately frozen at -80 C and thenkept at -20 C until analysis. To analyze the
TRAP and ALP activities, an aliquot of 100 μlof 20 mM para-nitrophenyl-phosphate in anacid buffer (0.1 M sodium acetate, including
20 mM tartrate, pH 5.3) or an alkaline buffer(100 mM Tris-HCl, pH 9.5; 1 mM MgCl2) was
added to each microplate well with a meltedacid buffer or alkaline buffer solution. This
plate was incubated at 20 C for 30 min while
being shaken. After incubation, the reaction
was stopped by adding 50 μl of a 3 N NaOH20 mM EDTA solution. A colored solution of
150 μl was transferred to a new plate, andthe absorbance was measured at 405 nm. The
absorbance was converted into the amount ofpara-nitrophenol (pNP) produced by using astandard curve for pNP. Detailed methods
described by Suzuki et al. (2009b).

performance liquid chromatograph equippedwith a fluorescence detector. The analyte wasseparated on a column (Discovery RP Amide
C16, 250 x 4.6 mm i.d., 5 μm; Sigma-Aldrich).
Isocratic elution was employed using 10 mM
phosphate buffer solution (pH 7.0)/
acetonitrile (55/45, v/v). The flow rate was
kept at 1.0 ml/min and the column
temperature was maintained at 40C. The
concentration of 4-OHBaA was quantified byusing deuterated 1-hydroxypyrene as aninternal standard. The detailed procedure is
described by Suzuki et al. (2015).

The mean for TRAP or ALP activity (8scales from one fish) in experimental group
was compared with that in control group.
Considering the variation among individuals,
the values indicates the ratio of the value ofeach time course for the initials value inrespective goldfish.

Figure 1 indicates plasma calcium levels after
BaA administration. BaA induced a significant
hypocalcemia at 24 (P < 0.01) and 48 h (P <0.05). Thereafter, plasma calcium levels
recovered at 72 h after injection of BaA.

Measurement of 4-OHBaA in the bile of BaAinjected goldfish:
At each time course, goldfish (n=5) wereanesthetized with ethyl 3-aminobenzoate,methanesulfonic acid salt (Sigma-Aldrich),and dissected. Then, bile was collected with asyringe from each gallbladder. The collectedbile was incubated with β-glucuronidase andarylsulfatase to hydrolyze BaA-glucuronide
and -sulphate, respectively. An aliquot of thesolution was injected into a high-Statistical analysis:
The statistical significance between thecontrol and experimental groups wasassessed by Student’s t-test. The selectedsignificance level was P < 0.05.
Results
Effects of BaA on plasma calcium levels in the
goldfish:Effects of BaA on osteoclastic and osteoblastic
activities in the scales of goldfish:
The results of osteoclastic activity are shownin Figure 2. BaA decreased osteoclastic
activity temporally. A significant decrease
between control group and experimentalgroup was obtained at 12 (P < 0.05) and 24 h
(P < 0.05). Thereafter, osteoclastic activity
was recovered towards control levels.
The response to BaA in osteoblasts was
slower than that in osteoclasts (Fig. 3). The
inhibitory effects (Fig. 3) were observed at 48(P < 0.01) and 72 h (P < 0.001).
75

Fig. 1. Effects of benz[a]anthracene (BaA) on plasma
calcium (Ca) levels in goldfish. Asterisk and double
asterisk indicate statistically significant differences at
P < 0.05 and P < 0.01, respectively, from the values in
the control goldfish.

Fig. 2. Effects of benz[a]anthracene (BaA) on
osteoclastic activity in goldfish. The mean for tartrateresistant acid phosphatase (TRAP) (8 scales from onefish) in experimental group was compared with that incontrol group. The values indicate the ratio of thevalue of each time course for the initial value inrespective goldfish (5 individuals for each timecourse). Asterisk indicates statistically significant
differences at P < 0.05 from the values in the control
goldfish.

Changes in 4-OHBaA in the bile of BaA-injected
goldfish:
4-OHBaA was detected in the bile of goldfish
after administration of BaA. At 48 h after
BaA injection, 4-OHBaA levels was highest(Fig. 4). In the bile of control goldfish,
4-OHBaA could not be detected.

Fig. 3. Effects of benz[a]anthracene (BaA) on
osteoblastic activity in goldfish. The mean foralkaline phosphatase (ALP) (8 scales from one fish)in experimental group was compared with that incontrol group. The values indicate the ratio of thevalue of each time course for the initials value inrespective goldfish (5 individuals for each timecourse). Double asterisk and triple asterisk indicate
statistically significant differences at P < 0.01 and P< 0.001, respectively, from the values in the control
goldfish.

Fig. 4. Changes in 4-hydroxybenz[a]anthracene (4OHBaA) of the bile of goldfish after benz[a]anthracene(BaA) injection. 4-OHBaA did not detect in the control
goldfish.

Discussion
We indicated that BaA (5µg/g body weight)(around 10-5 M) suppressed osteoclastic
activity at 12 and 24 h after injection.
Resulting from inhibition of osteoclasts in thescales of goldfish, plasma calcium levels
decreased. Therefore, we are the first to76

demonstrate that BaA directly influenceplasma calcium concentration. In ourprevious study, cadmium induced ahypocalcemia in goldfish (Suzuki et al.,2004b). Goldfish were kept in watercontaining cadmium (10 -7 M) for 2, 4, and 8
days and after 4 days of exposure, there wasnoticed a significant hypocalcemia (Suzuki etal., 2004b). Thereafter, plasma calcium levels
further decreased at 8 days after exposure to
cadmium (10-7 M) (Suzuki et al., 2004b). In
the present study, plasma calcium levels
recovered to control levels at 72 h due to theincreased osteoclast activity. In the case of
cadmium, however, plasma calcium levels
could not recover to control levels. Therefore,
toxicity of BaA seems to be less than that of
cadmium.
We detected 4-OHBaA in the bile ofgoldfish after administration of BaA. It is
possible that that metabolized 4-OHBaA
suppresses osteoclastic and osteoblastic
activities in the scales of goldfish. Suzuki et al.
(2009a) reported that 4-OHBaA inhibited
osteoclastic and osteoblastic activities in thecultured scales of goldfish and wrasse. After
short-term incubation (6 h), the sensitivity ofosteoclasts in the scales of both goldfish andwrasse was higher than that of osteoblasts inthe scales of both fish (Suzuki et al., 2009a).
Therefore, OHBaAs may firstly inhibit
osteoclastic activity and then suppress
osteoblastic activity. These phenomena are acause of the disruption of the bonemetabolism and the induction of spinal
deformities.
PAHs, such as benzo[a]pyrene, wereshown to inhibit osteogenesis in rat bonemarrow cells (Andreou et al., 2004).
Benzo[a]pyrene and 7,12-dimethylbenz[a]-anthracene have also been shown to inhibit
osteoclastogenesis in rabbit (Voronov et al.,2005). The present data are consistent withthese results. PAHs have been reported to beconverted into OHPAHs in the presence of
cytochrome P450 in human bone marrowcells (McCord et al., 1996). In mammals aswell as fish, therefore, we strongly suggestthat metabolized OHPAHs cause toxicity tobone tissue.
Teleost scales, like the mammalianendoskeleton, are known to work as apotential internal calcium reservoir (Suzuki
et al., 2008). We recently detected cathepsin
K and TRAP mRNA expression in scale
osteoclasts (Azuma et al., 2007). In
osteoblasts, we detected osteoblast-specificmarkers, such as runt-related transcription
factor 2, osterix, type 1 collagen, ALP, andosteocalcin (Thamamongood et al., 2012). It
has also been demonstrated that theosteogenesis of regenerating scale is quite
similar to that of mammalian membranebone (Yoshikubo et al., 2005). Therefore, thefeatures of osteoclasts and osteoblasts inscales are similar to those found in mammals.
In addition, calcitonin, a hypocalcemic
hormone, directly suppressed osteoclastic
activity in normal scales of teleosts as well as
in mammalian bone (Suzuki et al., 2000). In
our experiments, osteoclasts were activatedby parathyroid hormone (a calciumregulating hormone) in goldfish scale as wellas in mammalian bone (Suzuki et al., 2011).
Also, the effects of endocrine disrupters, suchas bisphenol-A (Suzuki and Hattori, 2003)
and tributyltin (Suzuki et al., 2006), andheavy metals, i.e., cadmium and organic
mercury (Suzuki et al., 2004b), on osteoblasts
and osteoclasts have been examined using
the cultured goldfish scales. We indicated77that cadmium (even at 10 -13 M) had an effecton the osteoclastic activity in the scales ofgoldfish (Suzuki et al., 2004b). Moreover, we
indicated that seawater polluted with highly
concentrated PAHs inhibited osteoblastic
activity in the scales of goldfish even if
polluted seawater was directly added into
culture medium at dilution rates of 500 times(Suzuki et al., 2016). Thus, fish scale assay
seems to be very useful to evaluate the effectof environmental pollutants on the bonemetabolism.
Storm water runoff and atmosphericdeposition are now the largest sources ofaquatic PAHs contamination (Lima et al.,
2003; Li and Daler, 2004). In addition topollution from the atmosphere to the water,
accidental oil spills, such as those from the
Deepwater Horizon, the Exxon Valdez, andthe Nakhodka, directly caused PAH pollution
in a marine environment (Bue et al., 1998;
Heintz et al., 2000; Hayakawa et al., 2006; deSoysa et al., 2012). In the Nakhodka C-heavy
oil, 210 µg/g of benz[a]anthracene having
four aromatic rings was detected (Hayakawa
et al., 2006). This concentration is similar tothat used in the present experiment.
Immediately after an oil spill, the high level of
PAHs influenced marine animals, including
fish. For a long time (more than 14 years),furthermore, the toxicity of PAHs originatingfrom an oil spill affected many marine
animals (for a review, see Peterson et al.,2003). In teleosts, PAHs have reproductive(Hoffmann
and
Oris,
2006)
and
developmental toxicity (Barron et al., 2004;
Billiard et al., 2006). Also, immune toxicity
has been reported (for a review see, Reynaud
and Deschaux, 2006). Taking these facts
together with our study, much attention

should be given
contamination.

toaquatic

PAHs

Acknowledgments
This study was supported in part by grants to
N.S. (Grant-in-Aid for Scientific Research [C]No. 16K07871 by JSPS), to T.S. (Scientific
Research [C] No. 15K07126 by JSPS), and to
H.M. (Scientific Research [C] No. 15K11034
by JSPS). This work was performed under thecooperative research program of the Instituteof Nature and Environmental Technology,
Kanazawa University, Acceptance No. 17011,
in 2017.
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