In vitro genotoxicity tests for polyhydroxybutyrate – A synthetic biomaterial

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Toxicology in Vitro 22 (2008) 57–67 www.elsevier.com/locate/toxinvit

In vitro genotoxicity tests for polyhydroxybutyrate – A synthetic biomaterial Abdulaziz Qaid Ali, Thirumulu Ponnuraj Kannan *, Azlina Ahmad, Ab. Rani Samsudin School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia Received 22 April 2007; accepted 1 August 2007 Available online 17 August 2007

Abstract The aims of this study are to determine the mutagenicity of a locally produced polyhydroxybutyrate (PHB) using Salmonella mutagenicity test and to find out if PHB altered the expression of p53 and c-myc proto-oncogenes and bcl-xl and bcl-xs anti-apoptotic genes in the human fibroblast cell line, MRC-5. Different concentrations of PHB were incubated with special genotypic variants of Salmonella strains (TA1535, TA1537, TA1538, TA98 and TA100) carrying mutations in several genes both with and without metabolic activation (S9) and the test was assessed based on the number of revertant colonies. The average number of revertant colonies per plate treated with PHB was less than double as compared to that of negative control. For the gene expression analyses, fibroblast cell lines were treated with PHB at different concentrations and incubated for 1, 12, 24 and 48 h separately. The total RNA was isolated and analysed for the expression of p53, c-myc, bcl-xl and bcl-xs genes. The PHB did not show over or under expression of the genes studied. The above tests indicate that the locally produced PHB is non-genotoxic and does not alter the expression of the proto-oncogenes and anti-apoptotic genes considered in this study.  2007 Elsevier Ltd. All rights reserved. Keywords: Polyhydroxybutyrate; Salmonella test; Mutagenicity; Gene expression; p53; c-myc; bcl-xl; bcl-xs

1. Introduction In the medical area, a number of degradable polymers have been developed that break down in vivo into their respective monomers within weeks or a few months. Despite the availability of these synthetic degradable polymers, there is still a need to develop degradable polymers which can further extend the range of available properties, particularly mechanical properties. Polyhydroxyalkanoates are natural, thermoplastic polyesters and can be processed by traditional polymer techniques for use in an enormous variety of applications, including consumer packaging, disposable diaper linings and garbage bags, food and medical products. Polyhydroxybutyrate (PHB) was the first of the polyhydroxyalkaonates (PHAs) to be discovered and is also *

Corresponding author. Tel.: +609 7663684; fax: +609 7642026. E-mail address: [email protected] (T.P. Kannan).

0887-2333/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.08.001

the most widely studied and best characterized PHA. It is accumulated as a membrane enclosed inclusion in many bacteria to consist of up to 80% of the dry cell weight. When extracted, it has mechanical properties very similar to conventional plastics like polypropylene or polyethylene and can be extruded, moulded, spun into fibres, made into films and used to make heteropolymers with other synthetic polymers (Shilpi and Ashok, 2004). PHB belongs to the large family of PHAs that are common carbon energy storage materials produced by numerous bacterial species. PHB is non-toxic, insoluble in water and displays chemical and physical properties similar to polypropylene. PHB was discovered by Lemoigne in Bacillus megaterium (Lemoigne, 1926). In subsequent years, it was also found in other species of bacteria, where it acts as a source of carbon and energy. The chemical structure of PHB is –{O–CH(CH3)–CH2–(C@O)}n–. PHB is finding a useful application as an implant material due to its biocompatibility and resorbability (Lootz

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et al., 2001). PHB appears ideal for use as temporary stents, bone plates, patches, nails and screws (Malm et al., 1992; Peng et al., 1996). Saad et al. (1999) focussed on the compatibility of PHB to chondrocytes and osteoblasts isolated from male rats and reported that the osteoblasts have only limited ability to take up (phagocytose) PHB particles and there was no sign of any cellular damage. In order to investigate how cells recognize biomaterial, mRNA expression of oncogenes (bcl-xl, bcl-xs and cmyc) and tumor suppressor gene p53 in the cells that attached onto various polymeric surfaces was evaluated using the reverse transcription-polymerase chain reaction (RT-PCR) method (Shinya et al., 2000). An international standard lays down specific requirements for biocompatibility ISO 10993-3 (1982) including the tests based on the nature of the contact and the duration of implantation of the biomaterial. The standard stipulates that all materials that will be in contact with mucous membrane, bone or dentinal tissue where the contact exceeds 30 days, as well as all implantable devices where the contact exceeds 24 h, must undergo genotoxicity testing (Chauvel-lebret et al., 2001). Most tests included in this part of the international standard refer to the Organization for Economic Cooperation and Development (OECD) Guidelines for testing of chemicals (OECD, 1986). Based on the recommendations of the ISO 10993-3 (1982) and the ASTM (1987) standards, many different representative test methods for the determination of genotoxic and mutagenic effects of the synthetic implant material are applicable. These include mutagenesis and toxicity tests, chromosome aberration test and gene expression analysis. The aims of this study are to determine the mutagenicity of a locally produced polyhydroxybutyrate (School of Biological Sciences, Universiti Sains Malaysia, Malaysia) using Salmonella/microsome test and to find out if PHB altered the expression of p53 and c-myc proto-oncogenes and bcl-xl and bcl-xs anti-apoptotic genes in the human fibroblast cell line, MRC-5.

2. Materials and methods 2.1. The test biomaterial The biomaterial used in this study is a locally produced PHB by the School of Biological Sciences in the form of short solid fibres. PHB was produced by micro-organisms Ralstonia eutropha (formerly known as Alcaligenes eutrophus) that employ the polymer as a form of energy storage molecule to be metabolized when other common energy sources are not available (Sudesh et al., 2000). In order to produce PHB homopolymer, glucose was used as the sole carbon source in nitrogen-free mineral medium. PHB was purified from freeze dried cells by extraction with chloroform and subsequent precipitation in methanol. The purified PHB was then dried at room temperature for

extended period of time to remove all traces of solvents as per the method described by Lee et al. (2004). The concentrations of PHB used in these screening tests were selected based on the available cytotoxicity data. The highest concentration dose (5000 lg/plate) is limited by solubility which is recommended in the Ames test protocol (Hai et al., 2001; Neal and Walter, 1996). Test substances that are cytotoxic already below 5 mg/plate should be tested up to a cytotoxic concentration. Testing above the concentration of 5 mg/plate may be considered when evaluating substances containing substantial amounts of potentially mutagenic impurities (OECD, 1997). Hence, in this study, the maximum concentration of 5 mg/plate was used for all the tests. 2.2. Salmonella/microsome test (Ames test) The test was carried out at the highest dose of 5 mg/ plate as recommended (OECD, 1997 and Mortelmans and Zeiger, 2000) and the four lower of 2.5, 1.25, 0.625 and 0.3125 mg/plate were obtained by a dilution with a geometric progression of 2, i.e. replicating exactly the procedure carried out in the dose finding test. They were sterilized using an autoclave at 121 C for 20 min. 2.2.1. Tester strains Five tester strains were used in this study. The tester strains of the Salmonella typhimurium mutant strains (TA1535, TA1537, and TA1538) were obtained from American Type Culture Collection (ATCC), where as, the tester strains, namely TA98 and TA100 were obtained from Riken Com. Japan. The tester strains were checked for genetic integrity at the time of use and triplicate plates were used for each treatment level. They were tested on histidine dependence, biotin dependence, the combination of both, rfa marker (crystal violet) and the presence of the plasmid pKM101 (ampicillin resistance) (Mortelmans and Zeiger, 2000). 2.2.2. Negative and positive control substances The negative control used in this study was sterile distilled water. Specific positive controls were used in order to confirm the reversion properties and specificity of each tester strain and the efficacy of the metabolic activation system. Toxic positive control application caused genotoxic effect (reverse mutation) to bacterial strains. Four types of positive controls were used: sodium azide (NAN3) (CAS No. 195-11092, Wako Pure Chemical Industries, Japan) at a concentration of 5 lg/plate was used for strains (TA1535 and TA100), 9- aminoaridine hydrochloride monohydrate, 98% (CAS No. 52417-22-8, Acros Orgaics, USA) at a concentration of 50 lg/plate for TA1537 and 4-nitro-O-phenylenediamine, 98% (CAS No. 99-56-9, Acros Organics, USA) at a concentration of 2.5 lg/plate for strains (TA1538 and TA98) without metabolic activation system. The positive control 2-aminoanthracene (2AA) (CAS No. 017-06851, Wako Pure Chemical Indus-

A.Q. Ali et al. / Toxicology in Vitro 22 (2008) 57–67

tries, Japan) at a concentration of 5 lg/plate was used for all strains with metabolic activation system (Zhang et al., 2004). 2.2.3. Chemicals, reagents and medium The chemicals used in this study were citric acid (CAS No. 33114) obtained from Riedel-de Haem.GMBH.seelze, D-biotin (CAS No. 2031) obtained from Calbiochem, US, glucose-6-phosphate dehydrogenase (CAS No. G-5885) purchased from Sigma (USA), oxoid nutrient broth No.2 (CAS No. CM67) and oxoid agar (CAS No. CM3) purchased from OxoidLtd (England), BactoTM Agar (CAS No. 214010) obtained from BD, (France). L-histidine (CAS No. 1.04351.0100), sodium ammonium hydrogen phosphate tetrahydrate (CAS No. 1.06682.1000), magnesium sulfate (CAS No. 1.05886.1000), sodium dihydrogen phosphate monohydrate (CAS No. 1.06346.1000) and magnesium chloride anhydrous (CAS No. 8.14733.0100) were purchased from Merck (Darmstadt, Germany). Potassium chloride (CAS No. 7447-40-7), sodium phosphate monobasic hydrate (CAS No. 10049-21-5), sodium phosphate dibasic anhydrous (CAS No. 7558-79-4) were purchased from Mall (USA). Di-potassium hydrogen phosphate anhydrous (CAS No. 60355) was obtained from Fluka (Switzerland), D- glucose (dextrose) anhydrous (CAS No. 50-99-7) from Gibco (USA), ampicillin sodium salt (CAS No. 69-52-3) from Amresco (USA) and liver microsomal enzymes (S9 homogenate) (CAS No. S-2067) were purchased from Sigma (USA). 2.2.4. Standard plate incorporation assay Bacterial mutagenicity tests are generally conducted using one of two basic methods. In both these procedures, bacterial cultures are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method (Ames et al., 1975; Maron and Ames, 1983; Gatehouse et al., 1994), the components are combined in molten overlay agar and plated immediately onto minimal agar medium. In the pre-incubation method (Yahagi et al., 1975; Matsushima et al., 1980; Gatehouse et al., 1994) the treatment mixture is incubated and then mixed with the overlay agar before plating onto minimal agar medium. For both the techniques, after 2 or 3 days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates. 2.2.5. Experimental procedure To the sterile glass tubes, the following were added in sequence; 0.1 ml overnight culture of Salmonella strain, 0.1 ml of the test chemical dilution or sterile water and 0.5 ml of metabolic activation (S9) mix or sodium phosphate buffer (pH 7.4). The contents of the test tubes were then mixed, incubated for 20 min at 37 C. Then to each tube, 2 ml of molten top agar maintained at 43–48 C was added. The contents of test tubes were then mixed and poured onto the surface of GM agar plates. When

59

the top agar hardened, the plate was inverted and placed in a 37 C incubator for 48 h. The colonies are then counted by colony counter and the result was expressed as the number of revertant colonies per plate. For an agent to be considered mutagenic, it has to produce at least a two-fold concentration-dependent increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control (Neal and Walter, 1996). 2.3. Gene expression analyses 2.3.1. Cell line and cell culture Fibroblast cell line CCL-171 designated as MRC-5 (ATCC) was used in the present study. Commercial essential modified eagle medium (EMEM) (CAS No. 11095-080, Gibco, UK) was used. The complete media was prepared as follows: 89 ml of EMEM + 10 ml of fetal bovine serum + 1 ml of penicillin streptomycin + 1 ml of L-glutamine. As soon as MRC-5 cell line was received, it was added into culture flask containing 5 ml of the complete medium and the cell suspension was dispersed into single suspension by repeated gentle pipetting over the surface. When the cells became confluent, the cells were passaged many times prior to treatment with PHB. The cells were then treated with five doses of PHB (0.3125, 0.625, 1.25, 2.5 and 5 mg/ml) and incubated for (1, 12, 24 and 48 h) at 37 C in a CO2 incubator. 2.3.2. Total RNA isolation Total RNA was obtained from 1 · 107 cells using RNeasy kit (Qiagen). The RNA was quantified by measuring the absorbance at 260 nm. The purity of the RNAs was assessed by the 260/280 nm absorbance ratio. Total RNA was adjusted to a final concentration at 20 ng/ll. These samples were used for reverse transcriptase-polymerase chain reaction. 2.3.3. Reverse transcriptase-polymerase chain reaction (RTPCR) Polymerase chain reaction (PCR) amplification (final reaction volume 2 ll) with RNA (4 ll per sample) was performed with One-step RT-PCR Kit (CAS No. 210210, Qiagen). The template RNA, primer solutions, dNTP mix, 5· Qiagen one-step RT-PCR buffers and RNase–free water were thawed and placed on ice. The solutions were mixed completely before use to avoid localized differences in salt concentration. A master mix was prepared according to Table 1. The master mix was mixed thoroughly and 21 ll of it was dispensed into PCR tubes. The sequences of the primers were designed according to Cury-Boaventura et al. (2004). The primer sequences, annealing temperature, number of cycles and their respective PCR fragment lengths are shown in Table 2. The template RNA (4 ll) was added to the individual PCR tubes and the thermal cycler was programmed

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amplification the samples were stored overnight at 2– 8 C, or at 20 C for long-term storage.

Table 1 Reaction components for one-step RT-PCR Components

Volume/ reaction (ll)

Final concentration

Master mix RNase–free water 5· QIAGEN one-step RT-PCR buffer dNTP mix (containing 10 mM of each dNTP) Primer A Primer B QIAGEN one-step RT-PCR enzyme mix Template RNA Total volume

11 5

– 1·

1

400 lM of each dNTP

1.5 1.5 1

0.6 lM 0.6 lM –

4 25

20 ng/reaction –

according to the program outlined in Table 3. The program includes steps for both reverse transcription and PCR. A reverse-transcription reaction temperature was 50 C for 30 min. The PCR amplification segment was started with an initial heating step at 95 C for 15 min to activate HotStarTaq DNA polymerase. For maximum yield and specificity, temperatures and cycling times was optimized for each new target and primer pair according to Table 3. The housekeeping b-actin gene was used as reference. The RT-PCR program was started while PCR tubes are still on ice. Once the thermal cycler was heated, then the PCR tubes were placed in the thermal cycler and after

2.3.4. Gel electrophoresis The PCR products were analyzed on a 2% agarose gel (CAS No. V3121. Promega, USA). The gel was subjected to electrophoresis for 25 min at 150 V and the bands were observed. The b-actin, p53, c-myc, bcl-xl and bcl-xs amplified products were (545, 549, 455, 470 and 201 bp) nucleotides in length, respectively. The intensity of a band for each gene was quantified by the photographed gels with AlphaEaseFc (AlphaDigiDoc 120-RevB). 2.3.5. Statistical analysis Statistical analysis was carried out using SPSS software version 12.0.1 for Windows employing one-way ANOVA test. 3. Results 3.1. Ames test It was found that the strains (TA1535, TA1537, TA1538, TA98 and TA100) showed no growth on the histidine and biotin plates, whereas, all strains showed growth on biotin histidine plates. Also, for the presence of rfa marker all Salmonella strains showed a zone of growth inhibi-

Table 2 The primer sequences, annealing temperature, number of cycles and their PCR fragment lengths Target gene

Sense primer

Anti-sense primer

Annealing temperature (C)

PCR fragment lengths

No of cycles

p53

5 0 -CTTGCATTCTG GGACAGCCAA-3 0 5 0 -TACCCTCTCAAC GACAGCAGCT-3 0 5 0 -CATGGCAGCAG TAAAGCAAGC-3 0 5 0 -ATCCAAACTGC TGCTGTGGC-3 0 5 0 -GTGGGGCGCC CCAGGCACCA-3 0

5 0 -GCACAAACACG AACCTCAAAGC-3 0 5 0 -CTTGACATTCTC CTCGGTGTCC-3 0 5 0 -GGTCAGTGTCT GGTCATTTCCG-3 0 5 0 -GTTCGACTTTC TCTCCTACAAGC-3 0 5 0 -CTCCTTAATGTCA CGCACGATTTC-3 0

59

594

30

55.5

455

30

56

470

30

56.5

201

30

56

545

35

c-myc bcl-xl bcl-xs b-actin

Table 3 Thermal cycler conditions Target gene conditions

p53

c-myc

bcl-xl

bcl-xs

b-actin

Reverse transcription temperature (C) Reverse transcription time (min) Denaturation temperature (C) Denaturation time (min) Annealing temperature (C) Annealing time (s) Extension temperature (C) Extension time (min) Number of cycles Final extension temperature (C) Final extension time (min)

50 30 94 1 59 90 72 1 30 72 10

50 30 94 1 55.5 90 72 1 30 72 10

50 30 94 1 56 90 72 1 30 72 10

50 30 94 1 56.5 90 72 1 30 72 10

50 30 94 1 56 90 72 1 35 72 10

A.Q. Ali et al. / Toxicology in Vitro 22 (2008) 57–67

tion surrounding the disc of crystal violet. For the presence of plasmid pKM101 (ampicillin resistance), the growth was observed surrounding the disc of ampicillin with strains carrying plasmid pKM101 (TA100 and TA98). 3.1.1. Plate count of range finding tests In the preliminary experiment, the test on the biomaterial PHB was carried out at the highest dose of 5 mg/plate and at different concentrations of 0.3215, 0.625, 1.25 and 2.5 mg/plate for detection of cytotoxicity. There was no growth inhibition and the test plate showed no increase in the number of revertant colonies in all tester strains TA1535, TA1537, TA1538, TA98, and TA100 with biomaterial PHB, as well as the negative and positive controls using the pre-incubation method in the presence and absence of an exogenous metabolic activation system S9 mix (Tables 4 and 5). 3.1.2. Plate count of main tests For the plate count of main tests, tests were conducted similar to the plate count of range finding tests. In this study, PHB did not inhibit the growth of the testing organism, S. typhimurium at 5 mg per plate as well as at 0.3215, 0.625, 1.25 and 2.5 mg/plate. The results showed that PHB caused no mutation as no reversion was seen with any of the five bacterial tester strains either in the absence or presence of the S9 mix. The results showed that the number of revertants induced by the extracts of PHB, for all tester strains was close to those of the negative control (spontaneous rever-

61

tants, without extract) and was much lower than those of the positive control (with diagnostic mutagens) (Tables 6 and 7). Furthermore, to be considered mutagenic, the number of revertant colonies per plate containing the substance tested, must be at least more than twice the number of colonies induced by the solvent control. This was never seen in the present tests with PHB. In addition, no dose dependent effects have been demonstrated. The positive controls for each strain resulted in the expected increase in the number of revertant colonies, indicating the study was valid. 3.2. Gene expression analyses Fibroblast cell line (MRC-5) treated with PHB (0.3125, 0.625, 1.25, 2.5 and 5 mg/ml) for 1, 12, 24 and 48 h showed no increase in mRNA expression of p53, c-myc, bcl-xl, bclxs and b-actin as shown (Figs. 1–5). The PCR band intensities were expressed as O.D normalized for b-actin gene mRNA expression as shown in Tables 8–12. 4. Discussion 4.1. Polyhydroxyalkanoates (PHAs) PHAs are biodegradable plastics and are the only 100% biodegradable polymers. They are polyester of various hydroxyalkanoates which are synthesized by numerous micro-organisms as energy reserve materials when an essential nutrient such as nitrogen or phosphorus is available only in limiting concentrations in the presence of

Table 4 Results of range finding test for Salmonella typhimurium strains in the absence of S9 mix Treatment

PHB (mg/ml)

Negative control Positive control a PHB

0 0.0025–0.005 0.3215 0.625 1.25 2.5 5

Number of revertant (number of colonies/plate) TA1535

TA1537

TA1538

TA98

TA100

356 1006 273 295 176 161 258

243 770 186 193 210 243 273

105 576 24 48 73 81 108

200 816 136 131 230 163 240

263 703 163 213 171 200 175

a PHB (5 mg) was extracted in 1 ml sterile distilled water and extract was incubated for 24 h at 37 C and various aliquots (5, 2.5, 1.25, 0.625 and 0.3125 mg/ plate) were then tested for mutagenicity in the standard plate incorporation assay. The number of revertant colonies per plate is the mean value from triplicates obtained in one experiment.

Table 5 Results of range finding test for Salmonella typhimurium strains in the presence of S9 mix Treatment

PHB (mg/ml)

Number of revertant (number of colonies/plate) TA1535

TA1537

TA1538

TA98

TA100

Negative control Positive control PHB

0 0.0025–0.005 0.3215 0.625 1.25 2.5 5

296 683 316 273 305 286 326

363 1000 418 396 386 400 408

370 923 257 340 306 295 381

300 903 195 198 166 198 240

318 716 195 141 135 261 190

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Table 6 Mutagenicity test for negative control, positive control and PHB in main test for Salmonella typhimurium strains in the absence of S9 mix Treatment

PHB (mg/ml)

Number of revertant (number of colonies/plate) TA1535

TA1537

TA1538

TA98

TA100

Negative control Positive control PHB

0 0.0025–0.005 0.3215 0.625 1.25 2.5 5

318 716 195 141 135 261 190

293 864 230 203 240 290 296

147 655 28 56 96 105 144

245 890 179 190 260 193 285

313 853 230 260 223 243 213

Table 7 Mutagenicity test for negative control, positive control and PHB in main test for Salmonella typhimurium strains in the presence of S9 mix Treatment

PHB (mg/ml)

Number of revertant (number of colonies/plate) TA1535

TA1537

TA1538

TA98

TA100

Negative control Positive control PHB

0 0.0025–0.005 0.3215 0.625 1.25 2.5 5

366 779 363 300 346 334 393

415 1089 466 449 467 499 495

443 1020 341 398 368 383 447

340 1018 247 234 223 255 268

404 815 265 186 150 279 214

Fig. 1. Effect of PHB on p53 gene expression by RT-PCR. Lanes M: 100 base pairs DNA ladder. Lane 1: Control MRC-5 cell line without treatment. Lanes 2, 3, 4, and 5: 0.3125 mg PHB for (1, 12, 24 and 48 h). Lanes 6, 7, 8 and 9: 0.625 mg PHB for (1, 12, 24 and 48 h). Lanes 10, 11, 12 and 13: 1.25 mg PHB for (1, 12, 24 and 48 h). Lanes 14, 15, 16 and 17: 2.5 mg PHB for (1, 12, 24 and 48 h). Lanes 18, 19, 20 and 21: 5 mg PHB for (1, 12, 24 and 48 h).

Fig. 4. Effect of PHB on bcl-xs gene expression by RT-PCR. Lanes M: 100 base pairs DNA ladder. Lane 1: Control MRC-5 cell line without treatment. Lanes 2, 3, 4, and 5: 0.3125 mg PHB for (1, 12, 24 and 48 h). Lanes 6, 7, 8 and 9: 0.625 mg PHB for (1, 12, 24 and 48 h). Lanes 10, 11, 12 and 13: 1.25 mg PHB for (1, 12, 24 and 48 h). Lanes 14, 15, 16 and 17: 2.5 mg PHB for (1, 12, 24 and 48 h). Lanes 18, 19, 20 and 21: 5 mg PHB for (1, 12, 24 and 48 h).

Fig. 2. Effect of PHB on c-myc gene expression by RT-PCR. Lanes M: 100 base pairs DNA ladder. Lane 1: Control MRC-5 cell line without treatment. Lanes 2, 3, 4, and 5: 0.3125 mg PHB for (1, 12, 24 and 48 h). Lanes 6, 7, 8 and 9: 0.625 mg PHB for (1, 12, 24 and 48 h). Lanes 10, 11, 12 and 13: 1.25 mg PHB for (1, 12, 24 and 48 h). Lanes 14, 15, 16 and 17: 2.5 mg PHB for (1, 12, 24 and 48 h). Lanes 18, 19, 20 and 21: 5 mg PHB for (1, 12, 24 and 48 h).

Fig. 5. Effect of PHB on the house-keeping b-actin gene expression by RT-PCR. Lanes M: 100 base pairs DNA ladder. Lane 1: Control MRC-5 cell line without treatment. Lanes 2, 3, 4, and 5: 0.3125 mg PHB for (1, 12, 24 and 48 h). Lanes 6, 7, 8 and 9: 0.625 mg PHB for (1, 12, 24 and 48 h). Lanes 10, 11, 12 and 13: 1.25 mg PHB for (1, 12, 24 and 48 h). Lanes 14, 15, 16 and 17: 2.5 mg PHB for (1, 12, 24 and 48 h). Lanes 18,19, 20 and 21: 5 mg PHB for (1, 12, 24 and 48 h).

Fig. 3. Effect of PHB on bcl-xl gene expression by RT-PCR. Lanes M: 100 base pairs DNA ladder. Lane 1: Control MRC-5 cell line without treatment. Lanes 2, 3, 4, and 5: 0.3125 mg PHB for (1, 12, 24 and 48 h). Lanes 6, 7, 8 and 9: 0.625 mg PHB for (1, 12, 24 and 48 h). Lanes 10, 11, 12 and 13: 1.25 mg PHB for (1, 12, 24 and 48 h). Lanes 14, 15, 16 and 17: 2.5 mg PHB for (1, 12, 24 and 48 h). Lanes 18, 19, 20 and 21: 5 mg PHB for (1, 12, 24 and 48 h).

excess carbon source. They possess properties similar to various synthetic thermoplastics like polypropylene and hence can be used in their place. PHB belongs to a class of polyesters of 3-hydroxy acids that are synthesized in various bacterial genera (Hai et al., 2001). Wang et al. (2003) in their study carried out on mouse fibroblast cell line L929, found that an appropriate combination of hydrophilicity and hydrophobicity was important for the cell growth on poly(hydroxybutyrate-co-hydroxyhexanoate) PHBHHx

A.Q. Ali et al. / Toxicology in Vitro 22 (2008) 57–67

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Table 8 IDV for mRNA expression of p53 gene in MRC-5 cell line

Table 12 IDV for mRNA expression of b-actin gene in MRC-5 cell line

Test material

Test material

IDV Incubation time in hour (s)

NC C1 C2 C3 C4 C5

Incubation time in hour (s)

1

12

24

48

P value

36,240 36,230 36,250 36,295 36,232 36,230

36,160 36,170 36,200 36,210 36,160 36,160

36,120 36,138 36,170 36,180 36,122 36,114

36,090 36,100 36,140 36,160 36,110 36,120

0.428

NC C1 C2 C3 C4 C5

1

12

24

48

P value

28,246 28,230 28,256 28,290 28,233 28,230

28,163 28,170 28,205 28,211 28,160 28,161

28,121 28,138 28,170 28,181 28,123 28,112

28,093 28,103 28,140 28,160 28,110 28,122

0.411

Note: IDV: Integrated Density Value; NC: Negative control (MRC-5 cell line without treatment). C1: 0.3125 mg/ml of PHB per flask; C2: 0.635 mg/ml of PHB per flask. C3: 1.25 mg/ml of PHB per flask; C4: 2.5 mg/ml of PHB per flask. C5: 5 mg/ml of PHB per flask.

Table 9 IDV for mRNA expression of c-myc gene in MRC-5 cell line Test material

IDV

IDV Incubation time in hour (s)

NC C1 C2 C3 C4 C5

1

12

24

48

P value

33,268 33,226 33,209 33,190 33,202 33,270

33,260 33,221 33,221 33,197 33,196 33,261

33,176 33,144 33,152 33,110 33,119 33,198

33,200 33,154 33,159 33,120 33,140 33,193

0.151

Table 10 IDV for mRNA expression of bcl-xl gene in MRC-5 Test material

IDV Incubation time in hour (s)

NC C1 C2 C3 C4 C5

1

12

24

48

P value

31,370 31,383 31,423 31,430 31,393 31,373

31,230 31,251 31,296 31,301 31,254 31,376

31,290 31,318 31,350 31,355 31,335 31,376

31,358 31,378 31,443 31,443 31,393 31,376

0.525

Table 11 IDV for mRNA expression of bcl-xs gene in MRC-5 cell line Test material

IDV Incubation time in hour (s)

NC C1 C2 C3 C4 C5

1

12

24

48

P value

33,246 33,230 33,256 33,290 33,233 33,230

33,163 33,170 33,205 33,211 33,160 33,161

33,121 33,138 33,170 33,181 33,123 33,112

33,093 33,103 33,140 33,160 33,110 33,122

0.555

and PHB and implied that this may instructive significance for biomaterial selection and design. In the present study, we report the non-genotoxic potential of polyhydroxybutyrate (produced in short solid fibre form) and manufactured by School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia,

using a metabolic Salmonella/microsome assay and gene expression analyses. A preliminary study was conducted by Raouf et al. (2004), on this locally produced PHB on fibroblast cell line (MRC5) and found that the percentage of cells killed were 0.1% after 24 h and 3.8% after 7 days of incubation, indicating that PHB had low toxic effect. Prior studies by others in this area had been primarily restricted to two commercially available PHA polymers, namely, polyhydroxybutyrate and polyhydroxybutyrateco-valerate (PHBV). These materials have been evaluated for a variety of medical applications, which include controlled release, surgical sutures, wound dressings, lubricating powders, orthopedic uses (Hocking and Marchessault, 1994) and as a pericardial substitute (Duvernoy et al., 1995). Deng et al. (2002) investigated the polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyhexanoate) for possible application as a matrix for the three-dimensional growth of chondrocyte culture. Simon et al. (1999) reported on the use of polyhydroxyalkanoate as a biopolymer scaffold in tissue engineering application. Knowles et al. (1992) and Knowles (1993) reported that hydroxyapatite/PHB composite have favorable bioactive properties and therefore could be potentially considered for application as a degradable bone substitute material. In addition, the mechanical properties of the composite were found to be much closer to the properties of cortical bone (Doyle et al., 1991). Unverdorben et al. (2002) implanted PHB biodegradable stents into the iliac arteries of New Zealand white rabbits and reported that PHB instigated intense inflammatory and proliferative reactions with an increase in collagen, thrombosis and in-stent lumen narrowing which ban them from clinical use. An integral part of current biocompatibility testing involves the demonstration of cell proliferation, which is usually taken as a sign of positive biocompatibility when the materials sustain or, even better, promote cell proliferation (Van Kooten et al., 2000).

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4.2. Ames test The Ames test is a biological assay developed by Ames and coworkers in the early 1970s to assess the mutagenic potential of chemical compounds and complex environmental mixtures. As cancer is often linked to DNA damage, the test also serves as a quick assay to estimate the carcinogenic potential of a compound as the standard tests for carcinogenicity done on rodents take years to complete and is expensive to do. The Salmonella/microsome mutagenicity test has been sufficiently developed and validated to be seriously considered for widespread use to detect the mutagens. The considerable evidence obtained using this test that with few exceptions, carcinogens are mutagens, supports the desirability of using this type of rapid and economical test system as a screening technique (Ames, 1971; McCann et al., 1975; Ames et al., 1975). The Ames test enables the detection of the potential of test substances to induce reverse mutation in the histidine gene of modified S. typhimurium strains (Gatehouse et al., 1994). Mutagenic substances can induce reversion in histidine-deficient strains, which are then able to grow and form colonies in a histidine-limited medium, whereas non-reversed strains cannot grow. 4.2.1. Tester strains A set of histidine-requiring strains is used for mutagenicity testing. Each tester strain contains a different type of mutation in the histidine operon (Ames et al., 1973). In addition to the histidine mutation, the standard tester strains contain other mutations that greatly increase their ability to detect mutagens. One mutation (rfa) causes partial loss of the lipolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules. The other mutation is deletion of a gene coding for the DNA excision repair system, resulting in greatly increased sensitivity in detecting many mutagens (Ames, 1971 and Ames et al., 1973). For these reasons, we chose five strains (TA1535, TA1537, TA1538, TA98 and TA100) to carry out this study. Different strains were used because specific mutations within the strain make them more sensitive to respond to different mutagens. 4.2.2. Negative and positive controls The negative control used in this study was sterile distilled water. Other solvents that may be considered are: acetone, ethylalcohol (95%), tetrahydrofuran, dimethylformamide and methylethylketone (MEK). These other solvents may be toxic to the bacteria at higher concentrations. The solvent of choice is sterile distilled water (Mortelmans and Zeiger, 2000). A positive control is a basic requirement in in vitro biomaterial testing. Application of toxic positive control caused reverse mutation to bacterial strains in expected pattern and thus our observation confirmed that the bacterial strains in this study were reliable from the physiological point of view. In each experiment, we routinely include positive mutagenesis control using

diagnostic mutagens to confirm the reversion properties of each strain. Positive controls using chemicals requiring metabolic activation confirm that the S9 mix is active (Ames et al., 1975). The results of positive control for each strain either in the presence or absence of S9 was increased more than or equal to two-fold compared to that of negative control. Similar results were found by Zhang et al. (2004), who used 2-nitroflurene at 1.0 lg per plate for TA98 and sodium azide at 2.0 lg per plate for TA100 without S9 activation and benzo[a] pyrene at 2.5 lg per plate for TA98 and 2aminoanthracene at 2.5 lg per plate for TA100 with S9 activation. These results indicate that PHB is devoid of mutagenic effect under the present test conditions on the five tester strains of S. typhimurium. Furthermore, to be considered mutagenic, the number of revertant colonies per plate containing the substance tested, must be at least more than twice the number of colonies induced by the solvent control. This was never seen in the present tests with the PHB. In addition, no dose dependent effects have been demonstrated. The positive controls for each strain resulted in the expected increase in the number of revertant colonies, indicating the study was valid. Similar to the results reported by Glosl et al. (2004), no mutagenicity was observed in the present study by Ames test either in the TA98 and TA100 strains or in the TA1535, TA1537 and TA1538 strains. PHB did not increase the number of revertants twice as the level of the spontaneous revertants, which is an important determinant for mutagenicity. Moreover, no significant dose response relationship was observed at different concentrations of PHB in all the strains treated with the positive control compound. Wening et al. (1995) reported that the test material, Kevlar49 as raw material and Kevlar49 extracts with ethanol or chloroform, did not exhibit mutagenic or cytotoxic activity by Ames test. Zinc oxide is reported not to elicit mutagenic effects on S. typhimurium TA98 and TA100 and magnesium oxide does not yield mutagenic effects on S. typhimurium TA97, TA100, and TAS102 in the presence or absence of a metabolically active microsomal fraction from rat liver S9 (Sawai et al., 1995). Zhang et al. (2004) cited that aristolochic acid was mutagenic to both TA98 and TA100 in the presence and absence of the S9 activation system. Although some previous reports indicated mutagenicity of Maillard reaction products without metabolic activation, no mutagenicity has been assessed with or without S9-pre-incubation. Yu et al. (2004) found that 1,5-anhydro-D-fructose at 5000 lg per plate induced no mutation as no reversion was seen with any of the five bacterial tester strains (TA98, TA100, TA102, TA1535 and TA1537) either in the presence or absence of activations system S9 mix. Ramires et al. (2005) reported no mutagenic potential of polyalkylimide hydrogel on the five tester strains (TA98, TA100, TA1535, TA1537 and TA1538) either in presence or absence of S9 activation system. Gahyva and Junior (2005) used S. typhimurium strains (TA98, TA97, TA100

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and TA102) to detect the mutagenic effect of endodontic substances and materials and reported that, no test substance or material exhibited mutagenic potential to any of the S. typhimurium strains used in their study. In this study, all the bacterial tester strains treated with PHB with and without S9 mix showed no increase of revertant colonies with increase in concentration of test biomaterial for both the range finding test and the main test. The number of revertant colonies was less than twice that of the solvent control for all the five bacterial tester strains. 4.3. Gene expression analyses In the present study, we used the QIAGEN one-step RT-PCR kit for amplification of RNA from cell lines according to the manufacturer’s instructions. This kit provides a convenient format for highly efficient and specific RT-PCR using any RNA. The kit contains optimized components that allow both reverse transcription and PCR amplification to take place in what is commonly referred to as a ‘‘one-step’’ reaction. Li and Mock (2005), used the QIAGEN one-step RT-PCR kit for amplification of viral target RNA. p53, because of its role in apoptosis, has earned the name ‘‘guardian of the genome.’’ It monitors the state of DNA and in the case of DNA damage, stalls the cell cycle (Martinez et al., 1991). In some systems, p53 seems to be important or essential for c-myc-evoked apoptosis. Expression of p53 appears to be associated with several, but not all, instances of apoptosis occurring after inappropriate cmyc over expression. Over expression of p53 by itself can cause apoptosis (Mihara et al., 2003). The proto-oncogene c-myc has been shown to play a pivotal role in growth control, differentiation and apoptosis. C-myc expression in proliferating normal cells is strictly dependent on mitogenic stimuli. Two lines of evidence indicate that, depending on the circumstances, both under and over expression of c-myc can lead to cell death (Listenberger et al., 2003). Besides bcl-2, the first protein to be identified with antiapoptotic potential (Tsujimoto et al., 1984), the bcl-2 family includes a number of proteins with either agonistic or antagonistic activity on bcl-2, whose relative amounts and interactions determine cellular susceptibility to programmed cell death (Sedlak et al., 1995). Among the best characterized proteins of this family are bax and bcl-x, with bcl-x possessing two different splicing isoforms, bcl-xl (long) which interacts with bcl-2 to inhibit programmed cell death and bcl-xs (short) which acts, similarly with bax, in pro-apoptotic activities (Cheng et al., 1996). Several studies have been performed to assess the role of alterations of bcl-2 family members in various malignancies: increased bcl-2 expression has been found in hematopoietic and solid tumors (Crawford et al., 1998; Hellemans et al., 1995; Pezzella et al., 1993; Silvestrini et al., 1994) and has been generally associated with a more favorable clinical outcome.

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In this study, there has been no over or under expression of p53, c-myc, bcl-xl and bcl-xs genes. These results indicate that there is no effect of PHB on mRNA expression of p53, c-myc, bcl-xl and bcl-xs genes. In a similar study, Cury-Boaventura et al. (2004), cited that oleic acid and linoleic acid on Jurkat cells caused over expression of p53 and c-myc. Huang et al. (2003), reported that dentin bonding agents rapidly induced c-jun and c-fos mRNA levels in human gingival fibroblasts cells, whereas, Schweikl et al. (1996) and Schweikl and Schmalz (1997), cited that dentin bonding agents containing glutaraldehyde are mutagenic as well as genotoxic agents. In the present study, the test biomaterial PHB did not increase the mRNA expression of either c-myc or p53, whereas, Xi et al. (2006), reported that medical polyacrylamide hydrogel (PAMG) increased the mRNA expression of c-myc, while the p53 and b-actin remained even. Chen and Huang (1998), reported that the level of c-myc mRNA was significantly reduced by curcumin (105–104 M) treatment and the level of bcl-2 mRNA was significantly reduced by 104 M curcumin. However, the alteration of the p53 mRNA level by curcumin (105–104 M) treatment did not achieve significance. Our findings in this study showed that PHB did not cause over or under expression of p53, c-myc, bcl-xl and bcl-xs genes on the fibroblast MRC-5 cell lines, indicating that PHB does not play a role in inducing apoptosis. In conclusion, all the bacterial tester strains treated with PHB with and without S9 mix showed no increase of revertant colonies with increase in concentration of test biomaterial for both the range finding test and the main test. The number of revertant colonies was less than twice that of the solvent control for all the five bacterial tester strains at all concentrations. PHB does not cause over or under expression of genes p53, c-myc, bcl-xl and bcl-xs in the fibroblast cell lines. The above tests indicate that the locally produced PHB is non-genotoxic under the present test conditions. Acknowledgements The authors are grateful to Dr. Sudesh Kumar, Ph.D., School of Biological Sciences for providing the test material, PHB and to the staff of Craniofacial Biology Laboratory, School of Dental Sciences and Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia for their assistance. References Ames, B.N., 1971. The detection of chemical mutagens with enteric bacteria. Chemical Mutagens: Principles and Methods for Their Detection. Hollaender Plenum Press, New York, pp. 267–282. Ames, B.N., Lee, F.D., Durston, W.E., 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proceedings of the National Academy of Sciences 70, 782– 786.

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