Silicon and iron levels in tissues of animals treated with a “ferrimagnetic ceramic” with oncotherapeutic potential (anti-tumor) value

3. Trace ELem. Med. Biol. Mot. 16, pp. 255-259 (2002) http://www.urba nfischer.de/journa[s/jtracee[m

© 2002 by Urban & Fischer Ver[ag

Silicon and iron levels in tissues of animals treated with a "ferrimagnetic ceramic" with oncotherapeutic potential (anti-tumor) value Teresa Almeida ~, Maria Etisa Soares 2, Jos6 Cavatheiro I and Maria de Lourdes Bastos 2,*

I INEB/FEUP 2 CEQUP, Toxicology Department, Faculty of Pharmacy, University of Porto, Porto, Portugal

Received July 2001 • Accepted February 2002

Abstract

The stability in a biological environment of an injectable cement with oncotherapeutic potential - consisting of a glass powder of SiO 2 (35.6%), CaO (42.4%), P~O 5 (17%), Na20 (5%) and 30% of its weight of Fe304 dissolved in (NH4)2HPO 4 plus NH~H2PO 4- was evaluated referring to the release of silicon and iron. The experimental model was the rat, and organs (liver, kidney, spleen, lung, heart, and brain) of the implanted and control animals were collected for quantification of these elements by electrothermal atomization atomic absorption spectrometry methods. In most of the analysed organs no significant difference in the contents of silicon and iron between the implanted and the control animals was found.

Key words: silicon, iron, ferrimagnetic ceramic, oncotherapy

Introduction

Tumor cells are very susceptible to heat as was consis- tently verified both in vitro (I) and in vivo (2, 3). When submitted to hyperthermia, the vascular network involving the solid tumors presents a differential response and is much more vulnerable than are the normal capiLLaries (4). In fact, proliferating neovascu[ature of tumor is more fragile and thermosensitive then quiescent mature vessels of normal tissues; tumor cell death may then occur direct- Ly due to thermal injury or indirectly as a result of damage to the vascuLature which Leads to ischemia and uLtimateLy to death of the dependent tumor cells (5). Several studies demonstrated that the destruction of the neopLasic vascu- Lar network elicits the death of the tumor tissue by hypox- ia, acidification and Leak of nutrients (6-8).

The high temperatures (understood to be higher than 44 °C) used in this thermotherapy presuppose a con-

*Correspondence to: Maria de Lourdes Bastos, CEQUP, Toxicolo- gy Department, Faculty of Pharmacy, University of Porto, Rua Aniba[ Cunha 104, 4050 Porto, Portugal

trolled and Localized heating. The most common tech- niques used up to now (total or partial immersion of the body, extra-corporal circulation, microwave, radio-fie- quency, ultrasounds, laser rays) present serious limita- tions because they give rise to the heating of great volumes of the body, disabling clinic applications over 43 °C.

CavaLheiro et aL (9) have developed a glass ceramic destined to be injected in the vicinity of solid tumors and to stay there, which works as a vehicle and support of ferrimagnetic particles enabling the localized heating of the tumor. This is possible by applying a high fre- quency external magnetic field which will heat the ferrimagnetic particles, and this treatment can be repeated without the inconvenience caused by other methodologies.

One of the characteristics required of this ceramic device consists in its inertness and resistance to biodegra- dation, that is, the material used must release neither par- tides nor soluble elements which could elicit a tecidu[ar reaction (10). Because the material used in the present study is mainly constituted by silicon oxide (Si02) and fer-

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256 T. Almeida el at.

ric oxide (Fe304), silicon and iron were quantified in the principal organs of implanted and control rats in order to evaluate an eventual releasing of these elements and its quantity. Calcium was not envisaged in this study because it is a predominant element in the biological milieu (11).

The elements were quantified by electrothermic atomization atomic absorption spectrometry. For silicon quantification, a method previously used in an interlaboratory study (12) was applied, after convenient adaptations. For iron, the whole procedure was implemented and validated: sample digestion, recovery and accuracy studies, limit of detection and lineafity range.

Materials and methods

Ferrimagnetic ceramic device A glass powder constituted by SiO z (35.6%), CaO (42.4%), P205 (17%) and Na20 (5%) was conveniently heated and added with 30% of its weight of Fe304 to obtain an injectable paste after dissolving in a (NH4)zHPO 4 plus NH4HzPO 4 solution. The paste obtained is fluid during some minutes, enabling the injection in the body and becomes solid in minutes at 37 °C.

In vivo assay Different volumes of the material solution (20, 30 and 40 pL, depending on the expected size of the solid tumors in the rat) were injected in the muscle of the hind paw of Wistar rats, in three groups of eight animals each. The material was left in the animals during ten days. After this period, the animals were sacrificed and several organs were collected (spleen, heart, brain, Liver, tung and kid- neys) to quantify the levels of silicon and iron. In parallel, a control group of animals submitted to the same diet reg- imen was constituted. At the end of the experiment the same organs were collected and the same elements were quantified to obtain the control values. Slices from the different organs were also prepared for histological analysis.

Samples for analysis The organs referred to above were collected and minced with polypropylene (PP) material previously rinsed with HNO 3 (:15%) and ultrapure water, packed in PP decontaminated tubes and dried in an oven at _+ 60 °C for several days. The dried samples were reduced to powder in a Teflon container, and about 100 pg transferred to another Teflon container, which, after addition of 1 mL of HNO 3 and 250 pL of H20 z, was closed for digestion in an oven at 85/90 °C for 5 h. The digested solution was transferred to a decontaminated tube and diluted to a convenient volume.

Reagents Standards were prepared daily from 1000 mg/L solutions (Tritisol, Merck) of iron (1II) in HNO 3 (0.2% v/v). ALl the acids used were of Suprapure grade (Merck).

The chemical modifier used in iron measurements was Mg(N03)2, Suprapure grade (Merck), prepared at convenient concentration in 0.2% v/v HNO 3.

Table 1. Instrumental conditions and graphite furnace programmes for quantification of silicon and iron in organs of rats implanted with the glass bioceramic.

Parameter silicon iron

Wavelength (nm) 251.6 248.3

Drying temperature (°C) i00 200 100 200 Ramp (s) 20 20 20 20 Hold (s) 20 20 20 20

Ashing temperature (°C) 1400 1400 Ramp (s) 30 30 Hold (s) 30 30

Atomization temperature (°C) 2500 2400 Ramp (s) 0 0 Hold (s) 4 4

Injection volume 15/10 of sample/modifier (pL) a

Inert gas argon Flow rate (mL/min) 300 Background correction deuterium arc HGA tubes with integrated platform Gas stop flow atomization step Measurement mode integrated absorbance

a For iron determination the autosamp[er was programmed to pipette sequentially 10 pL of the modifier solution and 15 pL of the digested sample/standard solution and dispense them together on the platform. Chemical modifier: 0.0i mg Mg(N03) 2. Silicon was evaluated without chemical modifier.

Quantification of silicon and iron Silicon and iron quantifications were carried out in a Perkin-Elmer HGA-700 furnace installed in a Model 1t00B spectrometer with deuterium arc background correction, equipped with an AS-60 autosampLer and an Epson EX-800 printer. The analyses were performed using Perkin-ELmer HGA Tubes with Integrated PLatform.

Instrumental conditions and furnace programmes for the determination of both elements are summarized in Table 1.

Validation of methodologies Previous implemented and validated methodologies for silicon measurement in biological samples (12) were applied in the present study.

For iron, the analytical conditions were established by using standard iron acid solutions and digested liver sample solutions. The calibration against acidified standard solutions was performed in the linear range from 0.80-50.0 pg /k To calculate the detection limit of the method, 20 determinations were carried out with an iron acid standard solution. The detection Limit is the value calculated as the concentration corresponding to three times the standard deviation (SD) of the background noise and was 0.80 IJg/L. The precision of the analytical method was evaluated by measuring 20 times the absorbance sig-

J. Trace Elem. Med. Biol. 1614 (2002)

Si and Fe levels in animaL tissues treated with a "ferrimagnetic ceramic" 2 5 7

na[s in the same digested liver sample. For evaluation of the precision of the over-all procedure, measurements of 20 different digested liver samples were performed. The values obtained (relative standard deviation) were 1.4% and 5.1% for the analytical and the over-all procedure, respectively. For recovery studies, three different concentrations of iron standard solutions (10, 25, and 50 pg/L) were added to the liver samples (six portions for each concentration), the spiked samples submitted to the over- all procedure and the metal quantif ied by the established conditions. The obtained recoveries were 94 _+ 2, 93 _+ 2, and 95 _+ 1°1o, respectively.

Results

Concentrations of siLicon and iron (mg/kg dried material) in the different analysed organs of implanted and control animals are summarized in Table 2 and 3, respectively. Data were analysed by one-way between-group analysis of variance (ANOVA), followed by the Scheff6 Test. ResuLts with p < 0.05 were considered as statisticaLly different.

Referring to silicon level contents, kidney, heart, lung, and liver are very similar and are the richest organs in this element. These findings are in accordance with those pub- [ished by Adler eta[ . (13) showing that in the rat kidney, lung and liver are the organs where silicon accumulates. Brain and spleen showed similar silicon contents, but lower than in the other organs analysed.

There was no signif icant difference between the values found for silicon in the organs of control animals and silicon in the organs of animals implanted with the glass bioceramic. Also, the silicon contents were not different among the organs of animals injected with three different volumes of the material solution.

As was expected, the iron content of the organs is much higher than that found for silicon. The descending order of the various organs in terms of their iron content was spleen, liver, lung, heart, kidney and brain. These levels are correlated with the biological functions of iron and its distr ibution in the organism. Again, no significant differences in the iron content were found in the analysed organs of implanted animals and in the organs of animals of the control group, except for the iron content in the

Table 2. Silicon contents (mean values _+ sd, mg/kg dried weight) in the different organs of the control animals and of the animals after application of the ferrimagnetic glass ceramic.

Organ

spleen Uver Lung kidney brain heart

Control 1.48 4- 0.33 3.04 4- %12 4.43 4- 1.88 5.15 ± 3.15 1.62 4- 0.36 (n : 6) (n = 5) (n = 5) (n = 5) (n = 5)

4.89 4- 1.66 (n = 5)

Group I (20 pL) 1.36 + 0.21 4.10 + 1.35 4.51 + 0.87 4.22 + 0.75 1.67 + 0.07 4.58 4- 0.42 (n = 8) (n = 7) (n = 7) (n = 7) (n = 8) (n = 7)

Group II (30 pL) 1.48 4- 0.24 4.19 + 1.41 4.80 4- 0.82 5.18 + 1.09 1.62 4- 0.19 4.77 4- 0.46 (n = 8) (n = 7) (n = 8) (n = 7) (n = 7) (n = 6)

Group II I (40 pL) 1.47 _ 0.29 3.83 ± 1.03 4.26 + 0.90 4.38 4- 0.81 1.63 4- 0.21 4.61 4- 0.52 (n = 8) (n = 7) (n = 8) (n = 7) (n = 7) (n = 6)

Table 3. Iron contents (mean values ± sd, mg/kg dried weight) in the different organs of the control animals and of the animals after application of the ferrimagnetic ceramic.

Organ

spleen liver Lung kidney brain heart

Control 1357 4- 315 649 + 84 688 ± 120 250 4- 44 107 4- 24 482 4- 78 (n=6 ) (n=6 ) (n=6) ( n : 6 ) (n=6 ) (n=6 )

Group I (20 pL) 1378 __. 60 723 + 99 649 ± 51 250 4. 15 100 4- 10 464 4- 66 (n = 8) (n = 7) (n = 7) (n = 7) (n = 8) (n = 8)

Group II (30 pL) 1627 ± 127 657 + 84 558 ± 87 240 + 28 104 + 20 467 4. 57 (n = 7) (n = 8) (n = 7) (n = 7) (n = 8) (n = 8)

Group II I (40 pL) 1555 + 121 975 _+ 171" 700 _+ 76 243 ± 17 114 ± 23 444 +_ 50 (n = 7) (n = 8) (n = 7) (n = 8) (n = 7) (n = 8)

* Different from control and group I and group II animals (p < 0.05).

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258 T. A[meida et at.

liver of the animals from the implanted group with the higher volume of the ceramic paste. In this group, the Liver concentrations were significantly higher (p < 0.05) than those in the control animals (975 + 171 versus 649 + 84 mg/kg, respectively) as well as of the animals injected with the other volumes of the ceramic paste (975 +_ 171 versus 723 _+ 99 and 657 + 84 mg/kg).

Discussion and conclusion

The biological significance of the elements investigated in this study must be separately considered. Silicon may be needed for normal development of the connective tissue (14) and is considered as a possibly essential element (15). Referring to iron, its biological importance is unquestionable, being involved in vital metabolic pro- cesses and playing an absolutely essential role in cell via- bility.

However, iron overload, specially in its free form, is very toxic, evoking neurological disorders (16), liver injury (17) and cardiomyopathy (18). For silica, inflamma- tory events in macrophages involving reactive oxygen species (ROS) are described. In vitro studies exposing macrophages to silica and antioxidants demonstrated the involvement of ROS in the expression of cytotoxicity mediated by oxidative stress (19). As silica and iron, besides calcium, are the principal elemental components of the developed ceramic, i t was our purpose to evaluate its stability in the biological environment by monitoring silicon and iron levels in the principal organs of the implanted rats.

The Literature refers that in the rat lung, Liver and kidney are the preferred storage organs for silicon (13). Thus, these organs were selected for monitoring silicon Levels. Referring to iron, Liver, kidney, heart and spleen are storage organs; because of its neurotoxic potential when at high Levels, brain was also included in the present study.

The methods used in the quantification of the elements were validated previously for silicon (12) and during the present study for iron. For this Last element, the method implemented shows very good precision, both for analytical and over-all procedures with coefficients of variation of 1.4% and 5.1%, respectively. The accuracy, evaluated by the standard addition method, is also very good, with recoveries better than 90% for all the added concentrations.

As i t can be observed in Table 2, among the several analysed organs, the richest in silicon are kidney, heart, lung and liver. Lower contents were found in spleen and brain. In the context of this study i t is outstanding that, for all the analysed organs, there are no significant differences between the concentrations of this element in the analysed organs of the animals submitted to the glass bioceramic material and those of the control animals. This means that, in the conditions of the assay, there is no release of silicon from the implanted ceramic. Considering the characteristics of the biological environment, ceramics can display several degrees of susceptibility, depending on their solubility. Having in mind the composition of this glass material, mainly oxide ceramics, i t is expected

that i t presents high inertness because its constituents are chemically very stable due to the very strong covalent and ionic bonds (10).

Referring to iron, spleen was by far the richest organ followed by Liver, Lung and heart. The statistical treatment of data showed that there are no differences in the content of iron in most of the analysed organs both from implanted and from control animals. The only exception is the significantly higher iron content in the Liver from the animals implanted with the higher volume of cement com- pared with the control and the other two implanted groups. Observing the content of iron in the liver of the animals implanted with 20, 30, and 40 pL of cement, 723 _+ 99, 657 + 84, and 975 + 171 mg/kg respectively, although the last being significantly higher than the oth- ers, the levels are not dose dependent. Also, i t is well known that Liver is one of the principal storage organ of iron in the organism and this high value can, at Least in part, be due to biological variability.

Besides the exception of the iron content in the liver, there were neither differences in the element content between control and implanted animals nor between animals implanted with different doses of cement for both measured elements.

In conclusion, the principal objective of the present study was attained by demonstrating that, in the experimental model used, the material of the bioceramic implant is stable. The release of silicon and iron, if any, is not significant as demonstrated by analysis of the principal organs. ALthough there is the uncertainty of extrapola- tion of these results to human beings, the material stud- ied seems to pose no additional health risk for patients to be implanted with the bioceramic, although clinical trials have to be performed.

Acknowledgements The authors from INEB are grateful for the financial support from the Praxis XXI programme for the development of the project "Ap[ica~o de cer~micos ferrimagn6ticos na destrui~o de teci- dos tumorais e estudo de mecanismos de apoptose e resposta imuno[6gica"

References

1. Rofstad EK, and Brustad T (1986) Primary and secondary cell death in human melanoma xenografts following hyperther- mic treatment. Cancer Res. 46:355-361

2. Nishimura Y, Shibamoto Y, Jo S, Akuta K, Hiraoka M, Taka- hashi M, and Abe M (1988) Relationship between heat- induced vascular damage and thermosensitivity in four mouse tumors. Cancer Res. 48:7226-7230

3. Brown SL, Hunt JW, and Hill RP (1992) Differential thermal sensitivity of turnout and normal tissue microvascu[ar response during hyperthermia. Int. J. Hyperthermia 8: 501-514

4. Dudar TE, and 3ain RK (1984) Differential response of normal and tumor microcircu[ation to hyperthermia. Cancer Res. 44: 605-612

5. Hill SA, Smith KA, and Denekamp J (1989) Reduced thermal sensitivity of the vascutature in a slowly growing tumour. lnt. J. Hyperthermia 5:359-370

3. Trace Etem. Med. Biol. 16/4 (2002)

Si and Fe levels in animal tissues treated with a "ferrimagnetic ceramic" 2 5 9

6. Bicher HI, Hetze[ FW, Sandhu TS, Frinak S, Vaupel P, O'Hara MD, and O'Brien T (1980) Effects of Hyperthermia on normal and tumor microenvironment. Radiology 137:523-530

7. Song CW, Kang MS, Rhee JG, and Levitt SH (1980) The effect of hyperthermia on vascular function, pH, and cell survivaL. Radiology 137:795-803

8. Emami B, and Song CW (1984) PhysioLogicaL mechanisms in hyperthermia: a review. Int. J. Radiation Onco[ogy BioL Phys. 10:289-295

9. Cavalheiro J, Vasconcelos M, Afonso A, and Branco R (!996) Ferrimagnetic and biocompatible cement for tumours thermotherapy. Bioceramics 9:243-246

10. Coury AJ, Levy RJ, McMi[lin CR, Pathak Y, Ratner BD, Schoen FJ, WiLLiams DF, and WilLiams RL (1996) Degradation of MateriaLs in the Biological Environment. In: Ratner BD, Hoffman AS, Schoen FJ, Lemons JE (Eds) Biomateria[s Sci- ence. Academic Press, San Diego, pp. 243-260.

11. WiLLiams DF, and WiLLiams RL (1996) Degradative Effects of the BioLogical Environment on MetaLs and Ceramics. In: Rat- ner BD, Hoffman AS, Schoen FJ, Lemons JE (Eds) Biomateri- a[s Science. Academic Press, San Diego, pp. 260-267

12. Van Dyck K, Robberecht H, Van Cauwenbergh R, Dee[stra H, Arnaud J, Wi[[emyns L, Benijts F, Centeno JA, TayLor H, Soares ME, Bastos ML, Ferreira M, D'Haese PC, Lamberts LV, Hoenig M, Knapp G, Lugowski SJ, Moens L, Riondato J, Van Grieken R, C[aes M, Verheyen R, CLement L, and Uytter-

hoeven M (2000) Spectrometric determination of silicon in food and bioLogicaL samples: an inter[aboratory triaL. J. Anal At. Spectrom. 15:735-741

13. Adler AJ, Etzion Z, and BerLyne GM (1986) Uptake, distribu- tion, and excretion of 31siLicon in normal rats. Am. J. Physi- ol. 251:E670-E673

14. Beattie JH, and Avene[[ A (1992) Trace element nutrition and bone metabolism. Nutrition Res. Rev. 5:167-188

15. Robinson CH, and Law[er MR (1982) Mineral ELements. In: Robinson CH, Law[er MR (Eds) Normal and Therapeutic Nutrition, 16 th edition. MacmiLLan PubLishing Co., Inc., New York, CoLLier MacmilLan PubLishers, London, pp. 131-160

16. Sorond FA, and Ratan RR (2000) Ironing-out mechanisms of neuronal injury under hypoxic-ischemic conditions and potential role of iron chelators as neuroprotective agents. Antioxid. Redox Signal 2:421-436

17. 8onkovsky HL, and Larnbrecht RW (2000) Iron-induced liver injury. C[in. Liver Dis. 4:409-429

]8. Bartfay WJ, and Bartfay E (2000) Iron-overLoad cardiomyopathy: evidence for a free radical-mediated mechanism of injury and dysfunction in a murine mode[. BioL Res. Nurs. 2:49-59

19. Kang JL, Pack IS, Lee HS, and Castranova V (2000) Enhance- ment of nuclear factor-KB activation and protein tyrosine phosphory[ation by a tyrosine phosphatase inhibitor, per- vanadate, involves reactive oxygen species in siLica-stimu- Lated macrophages. ToxicoLogy 151:81-89

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Documents

Silicon and iron levels in tissues of animals treated with a “ferrimagnetic ceramic” with oncotherapeutic potential (anti-tumor) value