Toxicokinetics and metabolisms of benzophenone-type UV filters in rats

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Toxicology 248 (2008) 89–95

Toxicokinetics and metabolisms of benzophenone-type UV filters in rats Hee-Kyung Jeon, Sailendra Nath Sarma, Youn-Jung Kim, Jae-Chun Ryu ∗ Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology (KIST), P.O. Box 131, Cheongryang, Seoul, Republic of Korea Received 29 November 2007; received in revised form 11 January 2008; accepted 7 February 2008 Available online 26 February 2008

Abstract Sunscreens containing UV filters are recommended to reduce damage caused by solar UV radiation. Recently, benzophenone (BP)-type UV filters have become widely used as UV stabilizers in skin-moisturizing products and sunscreen lotions; however, very little information is available regarding the potential harmful effects of prolonged exposure to these compounds. Therefore, we investigated the toxicokinetics and metabolism of BP-type UV filters in rats using gas chromatography–mass spectrometry (GC–MS). To examine the metabolism of BP-type UV filters, we analyzed the parent compounds BP and 2-hydroxy-4-methoxybenzophenone (HMB). In rats, BP was mainly converted to benzhydrol (BH) and 4-hydroxybenzophenone (HBP) (i.e., type A UV filters). In contrast, HMB was converted into at least three intermediates, including 2,4-dihydroxybenzophenone (DHB), which was formed via o-demethylation and subsequently converted into 2,3,4-trihydroxybenzophenone (THB), and 2,2 -dihydroxy-4-methoxybenzophenone (DHMB), which formed via the aromatic hydroxylation of HMB (i.e., type B UV filters). Next, the toxicokinetic curve for BP showed a peak concentration (Cmax ) of 2.06 ± 0.46 ␮g/ml at approximately 4 h after BP administration. After a single oral dose of HMB, the Cmax of HMB reached 21.21 ± 11.61 ␮g/ml within 3 h (Tmax ), and then declined rapidly compared to the kinetic curve of BP. The concentration of these metabolites in rat blood decreased much more slowly over time compared to the parent compounds. Thus, our results indicate that such metabolites might have more significant adverse effects than the parent compounds over the long term. © 2008 Published by Elsevier Ireland Ltd. Keywords: Benzophenone-type UV filters; Toxicokinetics; Metabolism; Rats; Gas chromatography–mass spectrometry

1. Introduction The most important property of the benzophenone (BP)-type ultraviolet (UV) filters is the ability to absorb and dissipate UV radiation. Exposing unprotected skin to UV light (particularly UVB, 290–320 nm) over an extended period of time promotes premature ageing of the skin and skin cancer. BPtype UV filters have been used to protect materials that are subject to discoloration or deterioration when exposed to sunlight. These BP-type UV filters have a broad absorption spectrum (200–350 nm) that is thought to protect against both UVA and UVB radiation (Kaidbey and Grange, 1987). The widespread use of BP-type UV filters as photostabilizers in cosmetics has led to the exposure of millions of consumers on a daily basis (Cosmetic Ingredient Review Panel, 1983). In addition, BP-type UV filters

∗

Corresponding author. Tel.: +82 2 958 5070; fax: +82 2 958 5059. E-mail addresses: [email protected], [email protected] (J.-C. Ryu).

0300-483X/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.tox.2008.02.009

are used in sunscreens to protect patients from drug-induced photosensitization, phototoxicity, or photoallergic reactions (FDA, 1978). Especially, BP is also used in non-alcoholic beverages, frozen dairy products, baked goods and soft candy (NAS/NRC, 1979). The Possible Average Daily Intake (PADI) calculated from these food categories is 0.33 mg/day, although this intake is a gross exaggeration. It is based on the assumption BP is used in all foods in each category and that average portions of food from all categories are eaten every day (NAS/NRC, 1979). The US Food and Drag Administration (FDA) has approved 2hydroxy-4-methoxybenzophenone (HMB) for use as an indirect food additive. It is permitted in the formulation of rigid acrylics and modified acrylic plastics which are components of single and repeated use of food contact surfaces (FDA, 1990). However, relatively little is known about the toxic effects of BP-type UV filters or their metabolites. HMB, also known as oxybenzone, is a mono-methoxylated and mono-hydroxylated derivative of BP (Sahral and Sharman, 1957). The US National Toxicology Program (NTP) conducted oral and dermal toxi-

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city studies for HMB in rats and mice and found that body weight gain decreased and relative liver weight increased after oral exposure in both species. In addition, 5% dietary HMB caused decreased sperm concentration in the caudal epididymis in both species (NTP, 1991). Although the parent compound, BP, was negative for genotoxic effects in a Salmonella mutagenesis assay (Mortelmans et al., 1986) and in a Escherichia coli pol A assay (Fluck et al., 1976), HMB showed mutagenic effects in Salmonella (Zeiger et al., 1987) and induced sister chromatid exchange and chromosomal aberrations in Chinese hamster ovary cells (French, 1992). Among the metabolites, 2,2 -dihydroxy-4-methoxybenzophenone (DHMB) was also mutagenic in the Salmonella assay (French, 1992). Recently, our group used a forward gene mutation assay to demonstrate that DHMB and 2,3,4-trihydroxybenzophenone (THB) have genotoxic effects in L5178Y cells (Jeon et al., 2007). As the BP-type UV filters have the possible adverse effects, it needs to understand the metabolic pattern and kinetic behavior of these compounds. Previous studies have investigated the metabolism of HMB in rats following oral administration (EI Dareer et al., 1986), but they gathered only limited data on the rates of absorption and elimination from plasma and the area under the plasma concentration–time curve (AUC). Although one aspect reported by NTP was persistence, these findings have never been verified. Based on this previous research, we investigated the toxicokinetic and metabolism of BP-type UV filters in rats using gas chromatography–mass spectrometry (GC–MS) to assess their

oral bioavailability and to lay groundwork for the evaluation of comparative toxicity. 2. Materials and methods 2.1. Chemicals BP was obtained from Sigma (St. Louis, MO, USA) and benzophenoned10 (BP-d10 ) as internal standard (I.S.) was supplied by Supelco (Bellefonte, PA, USA). Benzhydrol (BH), 4-hydroxybenzophenone (HBP), HMB, 2,4dihydroxybenzophenone (DHB), DHMB and THB were purchased from Aldrich (Milwaukee, WI, USA). Their structures and relevant physico-chemical properties are given in Table 1. To instrument analysis, stock standard solutions of seven BP-type UV filters were prepared in methanol containing 1000 mg/l of each compound. From these standards, working standard mixtures containing each compound at 1, 10 and 100 mg/l were prepared daily in methanol, and used to spike the blood samples. A 10 mg/l solution as I.S. was prepared with methanol. All standards and working solutions were stored in the dark at 4 ◦ C prior to use. The solvent used, acetone, ethyl acetate, acetonitrile and methanol were of the highest available purity and were obtained from J.T. Baker (Phillipsburg, NJ, USA). Hydrochloric acid and boric acid were obtained from Junsei Chemical Co. (Japan), and Na2 SO4 was supplied by J.T. Baker (Phillipsburg, NJ, USA). The derivatization reagent, Nmethyl-n-(trimethylsilyl)trifluoroacetamide (MSTFA) was obtained from Sigma (St. Louis, MO, USA). Corn oil was obtained from Sigma (St. Louis, MO, USA).

2.2. Animal treatments Male Sprague–Dawley rats at 8–10 weeks of age (approximately 300 ± 25 g) were obtained from Deahan Biolink Co., LTD. (Korea). Group of 7 rats per each chemical treatment had free access to water and a standard diet (Han Lim Lab.

Table 1 Structure and some physico-chemical properties of the test compounds Formula

Molecular mass

CAS number

b.p./m.p. (◦ C)

Log Kow a

Benzophenone (BP)

C13 H10 O

182.22

119-61-9

305/49

3.38

Benzhydrol (BH)

C13 H12 O

184.24

91-01-0

297-299/65-69

2.71

4-Hydroxybenzophenone (HBP)

C13 H10 O2

198.22

1137-42-4

150-160/132-135

3.07

2-Hydroxy-4-methoxy benzophenone (HMB)

C14 H12 O3

228.24

131-57-7

150-160/66

3.52

2,4-Dihydroxy benzophenone (DHB)

C13 H10 O3

214.22

131-56-6

194/144-145

2.96

2,2 -Dihydroxy-4-methoxy benzophenone (DHMB)

C14 H12 O4

244.24

131-53-3

170-175/68

3.82

2,3,4-Trihydroxy benzophenone (THB)

C13 H10 O4

230.22

1143-72-2

-/140-142

–

Compound

Chemical structure

Type A-UV filters

Type B-UV filters

a

Kow , octanol–water partition coefficient.

H.-K. Jeon et al. / Toxicology 248 (2008) 89–95 Animal Co. Korea) until 24 h prior to being used for experiments, at which time only food was removed, and were kept under standard conditions (21–23 ◦ C, 12-h light:12-h dark cycle, relative humidity 45–55%). Animals were orally administered BP and HMB (100 mg/kg body weight, bw), dissolved in corn oil. To obtain a homogeneous dosing solution, BP and HMB were ultra-sonicated in corn oil for 5 min (Branson 5510, Switzerland). The dosing volume applied was 4 ml/kg bw. Blood samples were collected from the rat femoral artery into the heparinized polyethylene tube (PE-50, Daejong Instrument Indu. Co., LTD.) at different time points up to 24 h after administration and immediately centrifuged at 9000 rpm for 15 min to separate plasma samples. The plasma samples obtained were stored at −70 ◦ C until analyzed.

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at three concentration levels, 0.5, 1 and 5 ␮g/ml by five replicate. Recoveries were measured by comparison of the peak area values of non-extracted standards versus extracted standards of spiked plasma at the same concentration. The intraday and inter-day accuracy and precision were validated by analyzing the plasma samples spiked with standard solution of 0.5, 1 and 5 ␮g/ml, and performed with five replicate on the same day as well as on 5 separate days.

2.5. Toxicokinetic parameters

Plasma samples (100 ␮l) were thawed at room temperature, added to an equal volume of acetonitrile (100 ␮l), mixed by vortexing, and centrifuged at 15,000 rpm for 5 min to pellet precipitated proteins. Aliquots of the supernatant were treated with 6N HCl (200 ␮l) to hydrolyze bound compounds at 100 ◦ C for 1 h. The samples were then adjusted to pH 8.5 with borate buffer after cooling. The deuterated internal standard (10 ppm × 20 ␮l; 200 ng) was added to each sample. The samples were extracted into ethyl acetate, the organic phase was evaporated for dryness, and the residue was derivatized with MSTFA (50 ␮l, 30 min at 80 ◦ C) for analysis by GC–MSD.

Toxicokinetic calculations were performed on each individual set of data using the pharmacokinetic calculation software WinNonlin Standard Edition Version 1.1 (Scientific Consulting, Apex, NC, USA) by non-compartmental method (Gabrielsson and Weiner, 1994). The following parameters were generated by the program: (i) biological half-life (t1/2 ), calculated from the slope of the terminal phase; (ii) area under the curve (AUC), where AUC was calculated to infinity according to the linear trapezoidal rule, AUC = AUC0−t + AUCt−inf. ; (iii) area under the moment curve (AUMC), where AUMC was calculated to infinity according to the linear trapezoidal rule; (iv) maximum plasma concentration (Cmax ); (v) time to maximum concentration (Tmax ); (vi) total body clearance (Cl), where clearance = dose/AUC; (vii) apparent volume of distribution calculated based on the terminal phase (Vz /F); and (viii) mean residence time (MRT), calculated using AUMC/AUC. The metabolite profiles of BP and HBP were also examined by using plasma samples collected form individual rats at various time points.

2.4. GC–MS analysis

3. Results

Analysis of BP-type UV filters was performed by using a GC–MSD (HP 6890 plus-HP 5973, Hewlett Packard, USA) method previously described (Jeon et al., 2006) with minor modification. GC–MS analysis was carried out Ultra 2 (5%-diphenyl-95% dimethylsiloxane) from Agilent Technologies (Palo Alto, CA, USA) equipped with a capillary column (30 m length × 0.2 mm i.d., 0.33 ␮m film thickness). The flow rate of helium (Shinyang Oxygen Inc., Seoul, Korea) as carrier gas was 1 ml/min. The injector temperature was set to 280 ◦ C and sample injection (1 ␮l) was in splitless mode. The GC column temperature was programmed from 100 ◦ C, ramped at 6 ◦ C/min to 210 ◦ C, ramped at 25 ◦ C/min to 290 ◦ C, and held for 4 min. The interface was kept at 280 ◦ C and mass spectra were obtained at 70 eV. Three representative fragment ions were selected from the full-scan mass spectrum of each compound to identify and quantify the response under SIM mode. The quantitative analysis was performed by I.S. method using the peak area ratios relative to the BP-d10 . The linearity of the calibration curves was prepared by triplicate analyses of plasma samples spiked with standard solution of BP-type UV filters to obtain the concentrations of 0.1, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 mg/l, with a fixed concentration (2 mg/l) of I.S. To further validate the extraction efficiency of the proposed method, recovery testing was performed

3.1. Identification of BP-type UV filters in rat blood using GC–MS

2.3. Preparation of rat blood samples

To detect and identify the various metabolite forms of BP-type UV filters, we used GC–MS to study the kinetic behavior of BP and HMB in rats after oral administration. Under our experimental conditions, the lower limit of detection was approximately 0.01 ␮g/ml in rat plasma samples at a S/N ratio of 3. Recovery of the test chemicals varied from 76 to 114% in blood samples [relative standard deviation (R.S.D.) < 10.34%] and fell well within the predefined acceptable limits (Table 2). The data for all analytes exhibited good linearity, and the R2 values were greater than 0.999 in rat plasma samples. As shown in Table 3, the overall mean precision, defined by the RSD, ranged from 0.27 to 9.87% within a given day and from 0.96 to 13.89% on 5 different days. Analytical accuracy, which was expressed as

Table 2 Recovery data for BP-type UV filters in rat bloods Spiked level (␮g/ml)

Values (%)

Type A-UV filters BP

BH

Type B-UV filters HBP

HMB

DHB

DHMB

THB

0.5

Mean S.D. R.S.D.

80.70 0.85 1.06

75.86 5.48 7.22

97.92 1.26 1.29

114.23 3.92 3.43

93.29 1.99 2.13

99.78 5.20 5.21

75.51 6.02 7.97

1

Mean S.D. R.S.D.

98.90 0.84 0.84

90.51 12.95 14.31

107.55 1.89 1.75

111.40 3.32 2.98

102.75 1.33 1.29

111.63 1.69 1.51

100.56 7.00 6.97

5

Mean S.D. R.S.D.

98.01 1.00 1.02

92.90 8.52 9.17

104.59 2.62 2.51

110.28 1.98 1.79

104.43 2.11 2.02

110.40 3.06 2.77

94.88 9.81 10.34

R.S.D. (relative standard deviation) = (S.D./mean) × 100. BP: benzophenone; HBP: 4-hydroxybenzophenone; BH: benzhydrol; HMB: 2-hydroxy-4-methoxybenzophenone; DHB: 2,4-dihydroxybenzophenone; DHMB: 2,2 -dihydroxy-4-methoxybenzophenone; THB: 2,3,4-trihydroxybenzophenone.

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Table 3 Intra-day and inter-day precision and accuracy of BP-type UV filters for the GC–MS method in bloods Compound

Intra-day

Inter-day

Concentration (␮g/ml)

R.S.D. (%)

Added

Found

BP

0.50 1.00 5.00

0.447 0.991 4.768

0.71 1.69 1.20

BH

0.50 1.00 5.00

0.436 0.990 4.842

HBP

0.50 1.00 5.00

RME (%)

Concentration (␮g/ml)

R.S.D. (%)

RME (%)

Added

Found

−10.54 −0.91 −4.64

0.50 1.00 5.00

0.443 0.999 4.832

5.52 0.96 1.87

−11.47 −0.06 −3.35

3.25 3.36 3.05

−12.84 −1.02 −3.17

0.50 1.00 5.00

0.465 0.976 5.378

3.48 4.18 3.48

−7.05 −2.36 7.56

0.450 0.996 5.042

3.58 3.01 3.43

−9.98 −0.38 0.85

0.50 1.00 5.00

0.472 1.012 4.785

2.16 2.88 4.05

−5.66 1.25 −4.31

0.50 1.00 5.00

0.486 0.992 5.047

5.38 2.92 5.30

−2.75 −0.78 0.93

0.50 1.00 5.00

0.506 0.969 4.823

1.97 2.48 4.61

1.17 −3.10 −3.54

0.50 1.00 5.00

0.427 0.985 4.967

4.02 2.84 2.97

−14.59 −1.53 −0.66

0.50 1.00 5.00

0.448 0.972 4.776

1.84 2.47 5.42

−10.39 −2.80 −4.48

0.50 1.00 5.00

0.495 1.000 5.191

3.82 3.17 3.04

−0.97 0.01 3.81

0.50 1.00 5.00

0.485 0.941 5.043

3.51 3.62 5.08

−2.97 −5.94 0.85

0.50 1.00 5.00

0.431 0.954 4.701

3.47 9.87 0.27

−13.87 −4.57 −5.98

0.50 1.00 5.00

0.448 0.970 5.117

13.89 2.08 4.53

−10.44 −2.99 2.35

Type A-UV filters

Type B-UV filters HMB

DHB

DHMB

THB

R.S.D. (relative standard deviation) = (S.D./mean) × 100, RME (relative mean error) = [(found − added)/added] × 100. BP: benzophenone; HBP: 4-hydroxybenzophenone; BH: benzhydrol; HMB: 2-hydroxy-4-methoxybenzophenone; DHB: 2,4-dihydroxybenzophenone; DHMB: 2,2 -dihydroxy-4-methoxybenzophenone; THB: 2,3,4-trihydroxybenzophenone.

the percent difference in the mean detected value compared to a known concentration, varied from −14.59 to 3.81% within 1 day and from −11.47 to 7.56% on different days. Thus, the intra- and inter-day accuracy and precision were within acceptable limits for the analysis of blood samples as part of a toxicokinetic study. The optimal GC–MS conditions for quantifying seven UV filters were those that provided high sensitivity and good resolution of the peaks. 3.2. Metabolism of BP-type UV filters in rats To detect and identify BP-type UV filter metabolites, we examined the toxicokinetics of two typical parent compounds, BP and HMB, in male Sprague–Dawley rats. The metabolism of BP-type UV filters was examined using individual and pooled blood samples collected at various time points. BP was analyzed as a typical type A UV filter. After oral administration, blood samples analyzed via GC–MS showed three peaks, including the parent peak and two major metabolites, identified as HBP and BH (Fig. 1). One of these metabolites was hydrolyzed with sulfatase, not ␤-glucuronidase, to yield free p-HBP. In contrast, BH was not hydrolyzed by sulfatase or

␤-glucuronidase, but instead underwent reduction of the keto group. HMB was analyzed as a typical type B UV filter. After oral administration, blood samples analyzed via GC–MS showed four peaks, including the parent compound and three metabolites. The two major metabolites were identified as DHB and DHMB. DHB is formed via O-dealkylation of the methoxy side chain on ring A of HMB, whereas DHMB is formed via the aromatic hydroxylation of ring B at the ortho position. A third metabolite, THB, was also present at low concentrations; this highly polar compound is formed from DHB via the aromatic hydroxylation of ring A at the meta position (Fig. 1). Several previous studies have reported that BP also metabolized into DHB in addition to 4,4 -dihydroxybenzophenone, 2,2 -dihydroxybenzophenone, 2-hydroxybenzophenone, and 3hydroxybenzophenone (Nakagawa et al., 2000), but these compounds were not detected in our study. 3.3. Toxicokinetics of BP-type UV filters and metabolites in rats Blood samples collected at various time points after oral administration were analyzed using GC–MS. Experimental

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Fig. 1. Proposed metabolic pathways of BP-type UV filters in rats. It shows the metabolic process of type A-UV filters and type B-UV filters. BP: benzophenone; HBP: 4-hydroxybenzophenone; BH: benzhydrol; HMB: 2-hydroxy-4-methoxybenzophenone; DHB: 2,4-dihydroxy benzophenone; DHMB: 2,2 -dihydroxy-4methoxybenzophenone; THB: 2,3,4-trihydroxy benzophenone.

results for the plasma concentrations of parent compounds and subsequent metabolites over time are shown in Fig. 2. These plots show that the metabolites had longer absorption phases than the parent compounds. The toxicokinetic parameters were obtained via non-compartmental analysis of the concentration–time curve. In blood samples from untreated rats (prior to administration of the test compounds), the concentration of parent compounds and all identified metabolites were below the limit of detection. After administration, the compounds were detectable up to 24 h after administration. The toxicokinetic curve for BP showed a peak concentration (Cmax ) of 2.06 ± 0.46 ␮g/ml at approximately 4 h after BP administration (dose, 100 mg/kg), after which the concentration of BP slowly declined. The elimination half-life (t1/2 ) of BP and the AUC were 19 h and 47.17 ± 5.52 ␮g/ml h, respectively. The concentration of HBP was lower compared to the parent compound and peaked within 4 h after BP administration (Fig. 2A, Table 4). After a single oral dose of HMB, the compound was quickly absorbed. The Cmax of HMB reached 21.21 ± 11.61 ␮g/ml within 3 h (Tmax ) after administration, and then declined rapidly compared to the toxicokinetic curve of BP. The t1/2 of HMB (4.6 h) was shorter than BP, and the AUC was 104.89 ± 23.82 ␮g/ml h. The concentration of the predominant metabolite, DHB, decreased slowly compared to HMB (Fig. 2B, Table 5). The concentrations of BH and THB were at the limit of detection in all samples analyzed and therefore could not be quantified.

In addition, we suspect that the formation of metabolites caused the AUC of BP (47.17 ± 5.52 ␮g/ml h) to be much less than that of HMB (104.89 ± 23.82 ␮g/ml h). Vz /F relates the amount of test compound in the body to the concentration of test compound in the blood. Clearance of the test compound occurs the amount of test compound eliminated per unit time, depends on the amount (concentration) of the test compound in the body compartment. These parameters have been described Table 4 Toxicokinetic parameters for typical chemical of type A-UV filters, i.e., benzophenone administration in rats Parameters a

AUC0→∞ (␮g/ml h) Tmax b (h) Cmax c (␮g/ml) t1/2 d (h) Vz /Fe (ml/kg) MRTf (h) Cl/Fg (ml/h/kg)

BP 47.17 3.83 2.06 19.28 53.50 27.40 2.20

± ± ± ± ± ± ±

Values are expressed as mean ± S.E. (n = 5). Animals were oral administered 100 mg/kg bw BP. a Area under the concentration–time curve from time zero to infinity. b Time to maximum concentration. c Maximum plasma concentration. d Half life. e Apparent volume of distribution. f Mean residence time. g Apparent clearance.

5.52 2.14 0.46 8.54 16.43 11.70 0.22

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in previous reports (e.g., Kadry et al., 1995). Due to the possible bioactivation of BP-type UV filters and the formation of metabolites with increased toxic effects, toxicokinetic considerations are important for reliable risk assessment and route-to-route extrapolations. In addition, the extent of metabolite formation and clearance is expected to differ widely between oral versus dermal exposure. Although dermal exposure is of more concern in humans applying sunscreens containing BP-type UV filters, oral administration is the most common route in toxicological studies on BP-type UV filters. 4. Discussion

Fig. 2. Plasma time courses for type A-UV filters (A) and type B-UV filters (B) in rats following oral administration of 100 mg/kg bw BP (n = 5) and HMB (n = 7), respectively. BP is the typical chemical of type A and, BH and HBP are the major metabolite forms of BP. HMB is the typical chemical of type B and, DHB, DHMB and THB are the major metabolite forms of HMB. BP: benzophenone; HBP: 4-hydroxybenzophenone; BH: benzhydrol; HMB: 2-hydroxy-4-methoxybenzophenone; DHB: 2,4dihydroxybenzophenone; DHMB: 2,2 -dihydroxy-4-methoxybenzophenone; THB: 2,3,4-trihydroxybenzophenone.

Table 5 Toxicokinetic parameters for typical chemical of type B-UV filters, i.e., 2hydroxy-4-methoxybenzophenone administration in rats Parameters a

AUC0→∞ (␮g/ml h) Tmax b (h) Cmax c (␮g/ml) t1/2 d (h) Vz /Fe (ml/kg) MRTf (h) Cl/Fg (ml/h/kg)

HMB 104.89 2.71 21.21 4.58 9.79 7.11 1.33

± ± ± ± ± ± ±

Values are expressed as mean ± S.E. (n = 7). Animals were oral administered 100 mg/kg bw HMB. a Area under the concentration–time curve from time zero to infinity. b Time to maximum concentration. c Maximum plasma concentration. d Half life. e Apparent volume of distribution. f Mean residence time. g Apparent clearance.

23.82 0.61 11.61 0.48 2.93 0.80 0.32

Our results demonstrate that orally administered BP and HMB are rapidly absorbed from the gastrointestinal tract. Structurally, BP and HMB are diphenyl ketones. The presence of two aromatic rings confers some degree of lipophilicity on the compound, allowing more rapid absorption from the gastrointestinal tract and more rapid disappearance (biphasic pattern) from the plasma. Disappearance from the plasma may be the result of redistribution to other compartments of the body. Previous studies regarding the tissue distribution of HMB demonstrated that the liver contained the highest amount of HMB, followed by the kidney (Kadry et al., 1995). After oral administration, the main route of HMB elimination over the 96h observation period was via the urine, followed by the feces. Over 75% of urinary excretion occurred in the first 6–12 h following HMB administration, whereas 90% of fecal excretion occurred in the first 24 h (Kadry et al., 1995). In a related study, the total amount of parent compound recovered from the urine and feces was less than 60% of the administered dose after 96 h (Okereke et al., 1993), indicating the formation of metabolites. These results were confirmed through GC–MS, which detected several peaks with retention times corresponding to those of the seven known UV filters. In our study, the toxicokinetic parameters of BP and HMB were only determined after acid hydrolysis. Acid hydrolysis is necessary because BP and HMB, which possess hydroxyl and ketone groups, may form hydrogen bonds with plasma macromolecules. In fact, protein binding has been demonstrated for many drugs and chemicals with similar functional groups (Bos et al., 1988; Zini et al., 1988). Moreover, the possible formation of the glucuronides of these metabolites was expected by previous studies (Kadry et al., 1995). A direct quantitation of the glucuronides of these compounds in plasma samples could not be performed since the glucuronides decomposed during the ethyl acetate extractions necessary for isolation from the blood matrix. Therefore, it was attempted to quantify unconjugated metabolites (free form) in blood, but results obtained were not well reproducible. This likely is due to the relatively harsh conditions required for extraction and the chemical reactivity of the acyl glucuronides (Spahn-Langguth and Benet, 1992) which may bind to nucleophiles in blood matrix. In addition, we used a single step liquid-phase extraction and GC–MS to examine the metabolism and kinetics of BP-type UV

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filters in rat blood. Although relatively simple, this method was both sensitive and reliable (i.e., precise and accurate) to study the metabolism and toxicokinetics of BP-type UV filters. In conclusion, BP was mainly metabolized to BH and HBP (type A UV filters), whereas HMB was enzymatically converted to at least three intermediates, including DHB, DHMB, and THB (type B UV filters). The concentration of these metabolites in rat blood decreased much more slowly over time compared to the parent compounds. Thus, it indicate that such metabolites might have more significant adverse effects than the parent compounds over the long term. This result can be lay groundwork for the evaluation of comparative toxicity of BP-type UV filters. Previous studies in humans have indicated that the dermal absorption of organic UV filters during the application of sunscreens is very limited; in fact, only very low concentrations of these UV filters were detected in the blood after repeated, intensive application (Lademann et al., 2005). However, further studies examining the toxicokinetics of BP-type UV filters and their metabolites in the blood after sunscreen application are required for a more conclusive toxicological assessment. Acknowledgements This work was supported by the intramural grants from Korea Institute of Science and Technology (KIST) and by the Research Foundation grants from Korea Ministry of Environment as “Eco-technopia 21 project” to Dr. Ryu J.C. of the Republic of Korea. References Bos, O.J., Remijn, J.P., Fischer, M.J., Wilting, J., Janssen, L.H., 1988. Location and characterization of warfarin binding site human serum albumin. Biochem. Pharmacol. 37, 3905–3909. Cosmetic Ingredient Review Panel, 1983. Final report on the safety assessment of the benzophenones-1, -3, -4, -5, -9 and -11. J. Am. Coll. Toxicol. 2, 35– 77. EI Dareer, S.M., Kalin, J.R., Tillery, K.F., Hill, D.L., 1986. Disposition of 2hydroxy-4-methoxylbenzophenone in rats dosed orally, intravenously, or topically. J. Toxicol. Environ. Health 19, 491–502. Fluck, E.R., Poirier, L.A., Ruelius, H.W., 1976. Evaluation of a DNA polymerase-deficient mutant of E. coli for the rapid detection of carcinogens. Chem. Biol. Interact. 15, 219–231. Food and Drug Administration, 1978. Report on sunscreen drug product for over the counter human drugs. Fed. Reg. 32, 412.

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