Assessment of the anticancer and antioxidant potentials of Spinosaurus tooth fossils: Application of energy dispersive X-ray fluorescence and field emission scanning electron microscopy techniques

INTRODUCTION

The fossil record provides an extraordinary archive of biological evolution, ancient ecosystems, and long-term geochemical transformation. Fossils are traditionally studied in the context of palaeontology, evolutionary biology, stratigraphy, and sedimentary geology; however, in recent years, fossilized materials have begun to attract attention in broader interdisciplinary fields, including biomaterials science, geochemistry, nanomedicine, and natural product-inspired therapeutic research. Fossilized hard tissues such as teeth, bones, shells, and mineralized plant remains are especially intriguing because they preserve not only morphological information, but also complex mineral signatures generated through long-term diagenetic processes. These mineral signatures may contain redox-active metals, apatite phases, silicates, carbon-bearing residues, and trace compounds capable of interacting with biological systems in unconventional ways.

Among the most iconic fossil vertebrates, Spinosaurus occupies a unique place in paleontological research. Spinosaurus was a giant theropod dinosaur that lived during the Late Cretaceous period, approximately 99–93.5 million years ago, in fluvial and coastal ecosystems of North Africa, particularly regions corresponding to present-day Morocco and Egypt.^[1,2] It is widely regarded as one of the largest carnivorous dinosaurs ever discovered and is distinguished by an elongated crocodile-like skull, conical non-serrated teeth, posteriorly positioned nostrils, and postcranial adaptations suggestive of semiaquatic or aquatic foraging behavior.^[3-6] Isolated Spinosaurus teeth are among the most frequently recovered fossils from the Kem-Kem Group of Morocco and related North African fossil-bearing deposits, and they have played a major role in reconstructing feeding ecology, habitat preference, and theropod diversity.^[7,8]

The tooth of Spinosaurus is of particular interest because of its distinctive structure and ecological function. Unlike the laterally compressed, blade-like teeth of many theropod dinosaurs, Spinosaurus teeth are generally conical, robust, and adapted for grasping slippery prey such as fish. During life, these teeth were composed of enamel, dentine, and internal structural tissues, but over millions of years, fossilization transformed them into mineralized geological specimens. The process of fossilization is not a passive preservation event; rather, it involves a series of physicochemical transformations such as permineralization, replacement, recrystallization, oxidation, and sedimentary mineral incorporation. As a result, fossilized teeth may retain a biomimetic architecture while also becoming enriched with diverse mineral phases and trace elements derived from the burial environment. The gross morphology of the authenticated fossilized Spinosaurus tooth used in the present study is shown in Figure 1. Representative binocular microscopic images of the fossilized Spinosaurus tooth are presented in Figure 2.

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Figure 1: Camera images of fossilized Spinosaurus tooth. Gross camera images showing the external morphology of the authenticated fossilized Spinosaurus tooth specimen used in the present study. The specimen displays a conical crown-like form, preserved external contours, and a mineralized fossil surface consistent with theropod dental architecture.

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Figure 2: Binocular microscopic images of fossilized Spinosaurus tooth obtained using EuroMaxAlpha. Binocular microscopic examination of the fossilized tooth showing fine surface texture, mineral deposition patterns, localized wear-related features, and irregularities in the fossil surface with magnification as 10x/2mm. These observations indicate that the specimen retains preserved morphological detail while also exhibiting secondary mineralization and surface alteration associated with fossilization.

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This geochemical transformation raises an important scientific question: can fossilized tooth material, after extensive diagenesis, display biologically relevant properties when processed as an extract or mineral preparation? Trace elements such as iron, manganese, magnesium, calcium, phosphorus, silicon, and associated oxygen-containing mineral phases are known to participate in redox reactions, catalytic behavior, ion exchange, and oxidative stress-related pathways in living systems. Iron and manganese, for example, can influence the generation of reactive oxygen species (ROS), while calcium-rich phosphate phases may affect cellular signaling and apoptosis-related pathways.^[9-15] From a biomaterials perspective, fossilized mineral composites could therefore serve as unusual natural matrices capable of interacting with cells through oxidative, ionic, or surface-mediated mechanisms.

Breast cancer remains one of the leading causes of cancer-related morbidity and mortality among women worldwide. Triple-negative breast cancer (TNBC) presents a major therapeutic challenge because it lacks estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, limiting the availability of targeted treatment options. The MDA-MB-231 human breast cancer cell line is a widely used in vitro model of aggressive and invasive TNBC and is frequently employed to screen novel compounds, natural products, metal-based agents, and biomaterials for anticancer potential. Cytotoxicity assays such as the 3-(4,5-diemethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay provide an initial measure of growth inhibition, while ROS-based assays offer mechanistic insight into oxidative stress-mediated pathways of cell injury and apoptosis.^[16-19]

ROS play a dual role in cancer biology. At controlled levels, ROS function as signaling molecules involved in proliferation, migration, and metabolic adaptation. However, excessive ROS accumulation can induce oxidative damage to DNA, proteins, and lipids, disrupt mitochondrial membrane potential, activate stress signaling cascades, and trigger apoptotic or necrotic cell death. Because many anticancer strategies rely on elevating ROS beyond the tolerance threshold of cancer cells, materials capable of modulating intracellular oxidative stress are of particular interest in cancer research.^[20,21]

In parallel with the biological significance of oxidative stress, the chemical complexity of fossil-derived materials warrants analytical investigation. Modern characterization tools such as Field emission scanning electron microscopy (FESEM), energy-dispersive spectroscopy (EDS), and energy-dispersive X-ray fluorescence (ED-XRF) allow detailed examination of fossil microstructure and elemental composition, while gas chromatography–mass spectrometry (GC-MS) can identify extractable organic and semi-volatile compounds associated with fossil matrices. These approaches provide an opportunity to connect paleontological specimens with biomaterial-like properties and to examine whether ancient, mineralized tissues may contain molecular or elemental features relevant to cellular bioactivity.

The present study was therefore designed to assess the anticancer and antioxidant-related potential of a fossilized Spinosaurus tooth specimen through an integrated analytical and biological workflow. The fossil was morphologically documented and characterized using FESEM, EDS, and ED-XRF to determine its microstructure and elemental composition. An ethanol–water extract of the powdered fossil was analyzed by GC-MS to identify putative bioactive constituents. The extract was then evaluated for cytotoxicity against MDA MB 231 human breast cancer cells using the MTT assay, and intracellular ROS generation was assessed by 2’,7’-dichlorodihydrofluorescein diacetate (DCFHDA) fluorescence staining. By combining palaeontology, geochemistry, analytical chemistry, and cancer cell biology, this study explores the possibility that fossilized Spinosaurus tooth material may represent a mineralized bioactive composite of interdisciplinary biomedical interest.

MATERIALS AND METHODS

FESEM imaging and EDS elemental analysis

FESEM was used to obtain high-resolution micrographs of the fossil surface. FESEM enabled examination of the microstructural organization of the fossilized tooth, including mineralized layers, surface threads, pores, and preserved textural features. Simultaneously, EDS was carried out to determine the elemental composition of selected regions of the fossil surface. The microstructural characteristics of the fossilized tooth observed by FESEM are shown in Figure 3.

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Figure 3: Field emission scanning electron microscopic images of fossilized Spinosaurus tooth. Representative field emission scanning electron microscopy micrographs showing the highly mineralized and structurally heterogeneous surface architecture of the fossilized tooth. The images reveal irregular layered deposits, thread-like mineral accretions, compact mineralized patches, and localized porous or fractured regions, indicating extensive diagenetic modification of the original dental tissue. The non-uniform surface morphology supports the interpretation that the specimen is a complex fossil biomineral composite rather than a simple preserved hard tissue. EHT: Electron high tension, WD: Working distance, Signal A, SE2: Secondary electron detector 2Mag: Magnification (e.g., 10 KX = 10,000×), CISL-AU: Laboratory/sample identification; Au most likely denotes gold-coated sample.

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RESULTS

FESEM-EDS elemental analysis

The FESEM-EDS spectrum demonstrated that the fossil surface contained a complex elemental composition. The upper tooth region was dominated by oxygen-rich mineral phases with additional contributions from carbon, iron, magnesium, manganese, silicon, aluminum, and apatite-associated mineral constituents.

The FESEM-EDS analysis of the fossilized Spinosaurus tooth demonstrated a predominantly oxygen-rich mineralized surface, with oxygen accounting for 69.16 wt% of the detected elemental composition. Carbon was the next most abundant element at 12.96 wt%, followed by iron at 7.39 wt%, while magnesium and manganese were present in smaller amounts at 0.49 wt% and 0.21 wt%, respectively. The high oxygen content is consistent with the presence of oxidized and phosphate-associated mineral phases within the fossil matrix, whereas the detection of iron, magnesium, and manganese indicates incorporation of trace metal-bearing mineral components during fossilization. Overall, the FESEM-EDS profile supports the view that the tooth surface has undergone substantial geochemical transformation and now exists as a heterogeneous biomineral composite enriched with both major and trace elements.

Calcium and phosphorus were also represented within the apatite-derived fossil matrix, while silicon and aluminum suggested silicified and sediment-associated mineral phases. Collectively, the FESEM EDS data indicate that the fossilized tooth surface is a chemically complex metal–mineral composite rather than a simple preserved hard tissue.

The spectrum demonstrates a highly mineralized and chemically heterogeneous fossil surface dominated by oxygen, with additional contributions from carbon, iron, magnesium, and manganese. Calcium and phosphorus indicate apatite-associated mineral phases, whereas silicon and aluminum suggest sediment-derived and silicified components incorporated during fossilization. The elemental profile supports substantial diagenetic alteration of the original tooth tissue. The corresponding FESEM–EDS spectrum is presented in Figure 4. The quantitative elemental composition determined by FESEM–EDS is summarized in Table 1.

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Figure 4: Field emission scanning electron microscopy-energy-dispersive spectroscopy (FESEMEDS) spectrum of fossilized Spinosaurus tooth. FESEM–EDS spectrum obtained from the fossilized Spinosaurus tooth showing a chemically complex and highly mineralized surface composition. Oxygen was the dominant detected element (69.16 wt%), followed by carbon (12.96 wt%) and iron (7.39 wt%), with smaller contributions from magnesium (0.49 wt%) and manganese (0.21 wt%). Calcium and phosphorus were also represented within the apatite-derived mineral framework, whereas silicon and aluminium suggested silicified and sediment-associated components. The elemental profile supports substantial diagenetic transformation of the original tooth tissue.

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Table 1: Elemental detection of fossilized Spinosaurus tooth using FESEM EDS.

Element	Weight %	MDL	Atomic %	Error %	Net Int.	R	A	F
C K	12.96	0.46	18.27	11.89	129.74	0.9134	0.0678	1.0000
O K	69.16	0.12	73.19	9.07	5288.63	0.9234	0.2026	1.0000
Mg K	0.49	0.05	0.34	12.33	74.30	0.9381	0.3163	1.0045
Al K	0.88	0.04	0.55	8.19	172.89	0.9413	0.4527	1.0074
Si K	8.77	0.03	5.29	5.54	2089.67	0.9443	0.5774	1.0045
K K	0.13	0.04	0.06	17.82	23.32	0.9574	0.8874	1.0319
Mn K	0.21	0.07	0.06	22.27	19.09	0.9705	0.9800	1.0980
Fe K	7.39	0.08	2.24	2.59	605.60	0.9725	0.9847	1.0637

Surface elemental composition of the fossilized Spinosaurus tooth as determined by FESEM-EDS analysis. The table summarizes the major and minor elements detected from the analyzed tooth region and reflects the chemically heterogeneous, oxygen-rich, and apatite-associated mineral character of the fossil surface. FESEM EDS: Field emission scanning electron microscopy energy-dispersive spectroscopy, R: Range correction factor, A: Absorption correction factor, F: Fluorescence correction factor

DISCUSSION

The present study demonstrates that fossilized Spinosaurus tooth material is a chemically and structurally complex biomineral matrix with measurable in vitro anticancer and oxidative stress-related activity against MDA-MB-231 human breast cancer cells. This represents an unconventional and exploratory attempt to connect paleontological biomineral material with cancer-related cellular assays through an integrated workflow combining FESEM, EDS, ED-XRF, GC-MS, MTT cytotoxicity analysis, and intracellular ROS assessment.

A central finding of the present work is the highly mineralized nature of the fossilized tooth. FESEM-EDS and ED-XRF analyses demonstrated that the fossil matrix contains oxygen-rich phases together with calcium, phosphorus, silicon, iron, magnesium, manganese, carbon, and additional trace elements. This is significant because the biological effects of mineralized materials are often governed not only by their bulk chemistry but also by the redox behavior, ionic release potential, surface charge, porosity, and structural heterogeneity of their mineral phases. Fossilized tooth material is especially interesting in this regard because it originates from a biological template that has been extensively transformed by geological time, potentially yielding a natural hybrid of apatite-derived architecture and sediment-associated mineral chemistry. The bulk elemental profile obtained by ED-XRF analysis is shown in Figure 5, while the trace elemental composition is summarized in Table 4.

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Figure 5: Energy dispersive X-ray fluorescence (ED-XRF) peaks corresponding to elemental identification of fossilized Spinosaurus tooth. ED-XRF spectrum of powdered fossilized Spinosaurus tooth showing a mineral-rich bulk elemental profile. The major peaks correspond to calcium and phosphorus, indicating that apatite-derived mineral phases remain dominant components of the fossil matrix. Additional peaks attributable to iron, magnesium, manganese, and silicon confirm the incorporation of trace metals and sediment-associated mineral constituents during fossilization. The spectrum complements the field emission scanning electron microscopy-energy-dispersive spectroscopy findings by demonstrating that both the surface and the bulk fossil material retain a chemically heterogeneous biomineral composition.

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Iron appears to be one of the most relevant elements in the context of the observed biological activity. Iron is a redox-active metal capable of cycling between Fe²⁺ and Fe³⁺ states and participating in Fenton-type reactions that generate hydroxyl radicals and other ROS.^[9,10] In cancer cells, which often already operate under elevated oxidative stress, further ROS generation can overwhelm antioxidant defenses and trigger cell death. Thus, the presence of iron-rich mineral phases in the fossilized tooth may contribute to oxidative stress induction following cellular exposure to the fossil extract or its released components. Similarly, manganese-containing phases may also influence ROS biology and mitochondrial function, as manganese is known to participate in oxidative stress regulation and superoxide dismutase-associated pathways.^[11]

Calcium and phosphorus are also important components of the fossil matrix. Phosphorus also warrants separate consideration because, within fossilized vertebrate hard tissues, it is retained predominantly as phosphate in apatite-derived mineral phases rather than as a free elemental constituent. Beyond its structural role, phosphate is relevant to mineral dissolution behavior, ionic exchange, membrane phospholipid organization, ATP-dependent metabolism, and phosphorylation-mediated cellular signaling. Accordingly, a phosphate-rich fossil biomineral may not be biologically inert, but instead may contribute indirectly to cell–material interactions and stress-related responses through its physicochemical behavior. The GC–MS chromatogram of the fossil extract is shown in Table 5.

In fossilized vertebrate hard tissues, these elements are commonly associated with apatite-derived mineral phases that originate from the original tooth or bone structure. Calcium phosphate-based materials have long been studied in biomaterials science because of their biocompatibility, ion exchange properties, and ability to interact with cellular membranes and intracellular signaling pathways. Disturbance of calcium homeostasis can activate endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis-related cascades in cancer cells.^[12,13] Magnesium, another detected element, may influence mineral lattice organization, electrostatic interactions, and membrane-level responses.^[14] Silicon-bearing phases may further contribute to the surface complexity and physicochemical behavior of the fossil matrix.

The MTT assay results showed a concentration-dependent cytotoxic effect of the fossil extract on MDA-MB-231 cells, with an IC₅₀ value of 72.67 µg/mL. This finding indicates that the fossil-derived preparation contains components capable of reducing cancer cell viability in vitro. While the exact potency cannot yet be directly compared with conventional chemotherapeutic agents or purified natural products, the result is notable because the test material was not designed pharmaceutical compound but rather an extract derived from a fossilized geological-biological specimen. The cytotoxicity findings suggest that the fossil matrix may contain either bioactive organic residues, redox-active mineral species, or a combination of both capable of interfering with cancer cell survival. The dose–response plot of the MTT assay optical density values is presented in Figure 6. The raw optical density values obtained from the MTT assay are summarized in Table 2.

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Figure 6: GraphPad Prism plot of optical density values of fossil extract at different concentrations. Dose–response plot derived from 3-(4,5-diemethylthiazol-2-yl)-2,5-diphenyltetrazolium assay optical density readings obtained at 570 nm after treatment of MDA-MB-231 human breast cancer cells with different concentrations of fossilized Spinosaurus tooth extract. There is graph shows progressive reduction in absorbance with increasing extract concentration, consistent with a concentration-dependent decrease in metabolically active viable cells.

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The ROS fluorescence assay provides mechanistic support for this interpretation. Treated MDA-MB-231 cells displayed increased DCFH-DA fluorescence compared with untreated controls, indicating elevated intracellular ROS levels. Excess ROS is known to damage nucleic acids, membrane lipids, proteins, and mitochondrial components, thereby promoting cell-cycle arrest, apoptosis, or necrotic pathways.^[20-22] Oxidative stress reflects a pathological imbalance between ROS generation and antioxidant defense, resulting in cumulative biomolecular damage and disease progression. Aruoma highlighted this redox disruption as a central mechanism in cancer and other chronic disorders, while underscoring the protective role of antioxidants in maintaining cellular integrity and reducing oxidative injury.^[22]

Cancer cells are particularly vulnerable to ROS overload because many malignancies maintain a high basal oxidative state due to rapid metabolism, mitochondrial stress, and oncogenic signaling. Therefore, materials that further elevate ROS may selectively push cancer cells beyond their oxidative tolerance threshold. The combined MTT and ROS findings in the present study suggest that oxidative stress-mediated cytotoxicity is a plausible component of the fossil extract’s biological effect. The concentration-dependent reduction in cell viability is illustrated in Figure 7, and the corresponding percentage cell viability values are summarized in Table 3. Representative microscopic images of untreated and treated MDA-MB-231 cells are shown in Figure 8.

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Figure 7: Cell viability (%) of fossil extract at different concentrations. Graph showing the percentage viability of MDAMB-231 cells following treatment with graded concentrations of fossilized Spinosaurus tooth extract. Cell viability was calculated relative to untreated control cells, which were taken as 100% viability. The reduction in viability with increasing concentration demonstrates a concentration-dependent cytotoxic effect of the fossil extract.

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Figure 8: Microscopic images of untreated control cells and treated cells at 500, 300, 100, 60, 20, and 10 µg/mL. Representative light microscopic images of MDA-MB-231 cells following exposure to different concentrations of fossilized Spinosaurus tooth extract. Untreated control cells show normal adherent morphology and confluence, whereas treated cells display progressive morphological alterations including reduced cell density, cell rounding, shrinkage, detachment, and loss of monolayer integrity, consistent with concentration-dependent cytotoxic stress (a-g) 100×.

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GC-MS profiling adds another important dimension to the interpretation of the results. The detection of anthraquinone-related molecules, pyrrole derivatives, sulfur-containing compounds, aromatic esters, piperazine-associated compounds, and iron-associated organometallic species suggests that the fossil extract may not behave as a purely inorganic mineral preparation. Anthraquinone derivatives are especially relevant because they are widely recognized in anticancer pharmacology and may act through DNA intercalation, redox cycling, topoisomerase inhibition, and apoptosis induction. Pyrrole and piperazine derivatives have also been reported in medicinal chemistry contexts for their influence on signaling pathways, metabolic activity, and drug-like interactions. Sulfur-containing compounds may modulate redox status and mitochondrial function, while aromatic ester-like molecules may reflect preserved or diagenetically modified organic components associated with the fossil matrix.

Taken together, the elemental and GC MS findings suggest that the biological activity of the fossilized Spinosaurus tooth may arise from a mineral–organic synergistic system rather than from a single isolated factor. This is an important conceptual point. Fossilized materials are not chemically homogeneous; they are products of long-term interaction between original biological tissues and surrounding geochemical environments. Consequently, any observed biological effect is likely to emerge from the combined influence of mineral phases, trace metals, preserved organics, sediment-derived compounds, and extraction-dependent constituents. The fossil extract studied here may therefore be considered a preliminary biomaterial-like composite rather than a conventional purified natural product.

From an interdisciplinary perspective, the study also highlights a broader conceptual bridge between palaeontology and biomedical materials research. Fossils are generally valued for the information they provide about ancient organisms and environments, but their mineralized composition may also make them relevant to geochemistry-driven biological investigation. This does not imply that fossils should be exploited indiscriminately as medicinal resources; fossil specimens are scientifically important and must be treated as part of geological and paleontological heritage. However, carefully designed laboratory studies on authenticated fossil fragments, powdered residues, or non-diagnostic materials may open new questions regarding ancient biomineral chemistry, redox-active geological matrices, and bioinspired mineral composites. The calculated IC₅₀ value and dose–response parameters are presented in Table 6. Representative intracellular ROS fluorescence images are presented in Figure 9. The quantitative analysis of intracellular ROS generation is shown in Figure 10.

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Figure 9: Intracellular reactive oxygen species (ROS) fluorescence images of untreated and treated MDA-MB-231 cells. Representative fluorescence micrographs of 2’,7’-dichlorodihydrofluorescein diacetate stained MDA-MB-231 cells showing intracellular reactive oxygen species generation in untreated control cells and cells treated with fossilized Spinosaurus tooth extract at 35.21 µg/mL. Compared with the untreated control, treated cells exhibited markedly stronger green fluorescence, indicating increased intracellular ROS accumulation and oxidative stress following exposure to the fossil extract. (a) Untreated control, (b) Treated - 35.21 µg/mL. (a and b) Magnification:100×, Scale bar:100 µm.

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Figure 10: Bar chart representing reactive oxygen species (ROS) levels in untreated and treated cells. Bar chart showing 2’,7’-dichlorodihydrofluorescein diacetate fluorescence based intracellular ROS levels in untreated control cells and cells treated with fossilized Spinosaurus tooth extract. ROS levels are expressed as relative fold change, with the untreated control normalized to 1.0. The elevated ROS level in treated cells supports the involvement of oxidative stress in the cytotoxic response induced by the fossil extract.

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The present study nevertheless has several limitations that must be acknowledged clearly. First, the biological evaluation was restricted to a single cancer cell line, MDA MB 231. No normal mammalian cell line was included to determine whether the fossil extract exhibits selective toxicity toward cancer cells. Second, the study did not quantify apoptosis markers such as caspase activation, Annexin V staining, mitochondrial membrane depolarization, or DNA fragmentation. Third, the antioxidant-related claim in the title should be interpreted carefully: although ROS modulation was assessed, the current data mainly support oxidative stress induction in cancer cells rather than classical antioxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); Ferric Reducing Antioxidant Power or related free-radical scavenging assays. If the journal or reviewers request stricter evidence for “antioxidant” activity, the title or discussion may need to clarify that the work addresses oxidative stress modulation rather than direct antioxidant capacity. Fourth, GC-MS identification of compounds in a fossil extract must be interpreted cautiously because contaminants, environmental organics, storage-associated compounds, or extraction artifacts cannot be ruled out without confirmatory analyses.

Future work should therefore focus on expanding the biological and chemical characterization of the fossil-derived material. Important next steps include testing against additional cancer cell lines and non-cancerous cells, evaluating apoptosis and cell-cycle markers, measuring mitochondrial membrane potential, quantifying oxidative stress biomarkers, and isolating active fractions through chromatographic purification. Advanced elemental mapping, X-ray diffraction, Fourier-transform infrared spectroscopy, inductively coupled plasma mass spectrometry, and liquid chromatography–mass spectrometry/MS could further clarify the mineral and molecular composition of the fossil matrix. If reproducible activity is confirmed, the fossilized Spinosaurus tooth may serve not only as a direct therapeutic material but as an inspiration for designing synthetic or bioinspired mineral composites that mimic its redox-active and biomineral characteristics.^[23]

A final conceptual point concerns the broader ecological and translational implications of fossil-bearing mineral environments. Fossil-rich terrains are often associated with mineral-enriched substrates, and such environments may influence the chemistry of surrounding sediments and potentially the phytochemical profile of medicinal plants growing nearby. Although this idea was not experimentally examined in the present study, it suggests an interesting future direction linking palaeontology, geochemistry, medicinal plant science, environmental biotechnology, and biomaterials research. Such ideas, however, must remain secondary to conservation priorities, and any future work in this area should be conducted within strict ethical and geological preservation frameworks.

Overall, the present results indicate that fossilized Spinosaurus tooth material is more than a paleontological curiosity. It is a diagenetically transformed mineralized composite with measurable in vitro biological effects and with sufficient chemical complexity to justify further investigation. The study does not claim that fossils are established anticancer agents, but it does propose that ancient biomineral systems may offer new inspiration for interdisciplinary research at the interface of geology, palaeontology, chemistry, and cancer biology.

CONCLUSION

The present study demonstrates that fossilized Spinosaurus tooth material possesses significance extending beyond conventional paleontological interpretation and may have preliminary relevance in anticancer and oxidative stress-related biomedical research. FESEM, EDS, and EDXRF analyses revealed that the fossilized tooth is a highly mineralized composite enriched with oxygen, calcium, phosphorus, silicon, iron, magnesium, manganese, carbon, and other trace elements. GC–MS profiling further identified multiple extractable compounds associated with chemical classes previously linked to oxidative stress modulation and cytotoxic activity.

Biological evaluation against MDA-MB-231 human breast cancer cells showed concentration-dependent cytotoxicity with an IC₅₀ value of 72.67 µg/mL, while intracellular ROS analysis demonstrated increased oxidative stress in treated cells. Together, these findings suggest that fossil-derived mineral–organic matrices may influence cancer cell viability through ROS-associated and apoptosis-related mechanisms.

Although the present findings remain preliminary and require additional mechanistic, toxicological, and in vivo validation, they establish an interdisciplinary basis for exploring fossil-derived biomineral materials in cancer-related research. The fossilized Spinosaurus tooth may therefore be viewed not only as a remnant of prehistoric life but also as a scientifically intriguing mineralized composite capable of inspiring future studies in geochemistry, biomaterials science, oxidative stress biology, and anticancer therapeutics.