Sociedade Brasileira de Dermatolodia Surgical & Cosmetic Dermatology


ISSN-e 1984-8773

Volume 3 Number 3

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Original Article

Biochemical and toxicological assessment of a Brazilian mineral water and its effects on the skin

Avaliação bioquímica e toxicológica de uma água mineral brasileira e seus efeitos cutâneos em uso tópico

Samanta Nunes1, Bhertha Miyuki Tamura1

Received on: 10/05/2011
Approved on: 30/08/2011

This study was carried out at the Instituto de
Bioengenharia da Pele - EVIC Brasil Ltda e
Tridskin Laboratórios Ltda.
Financial support: Trabalho recebeu material
do laboratório PuraInova para análise.

Conflicts of interests: Material fornecido pelo
laboratório PuraInova para realização do



Introduction:The French cosmetics industry sells spring mineral waters that are advertised as having biological benefits.
Objective: This study analyzes, in vitro and in vivo, a Brazilian mineral water's oligomin- erals composition, as well as its physical and chemical characteristics and biological effects.
Methods: Tests to evaluate physical chemical properties, cytotoxicity (viable cells) and irritability (het-cam test) were conducted. In vitro studies were performed to evaluate its capacity to induce the genic expression and immunohistochemical detection of filaggrin and aquaporin 3, nf-kb activity and fibroblast proliferation compared to Milli Q water.
Results: This water was found to be non-cytotoxic and non-irritating. In addition, it pre- sented a high content of strontium (0.61 mg/ml). The expression of filaggrin and its immunohistochemical tests were relevant. The aquaporin 3 increased 1.8 times and nf-kb decreased its activity by 47%. It was also capable of stimulating fibroblast proliferation.
Conclusion: The initial evaluation of the mineral water from Serra do Japi (SP, Brazil) indicates that it has the potential to be an adjuvant treatment in dermatology, since the results suggest it moisturizes the skin barrier, stimulates the proliferation of fibroblasts, and repairs and inhibits inflammatory reactions. Clinical studies should be done in order to reassure the in vitro results achieved on the present study.



A number of studies published in the literature have shown that spring mineral or thermal water have different physiochem- ical properties that can affect their clinical application in derma- tology. With plenty of hydrographic basins and mineral water springs, Brazil offers a sizeable opportunity to leverage its natu- ral resources; the waters should be analyzed and possible derma- tologic applications should be explored. This study describes the characteristics and biological effects of a Brazilian mineral water and clinical studies about its application on the skin. The min- eral water in question originates from pluvial waters infiltrated in the underground Alvorada spring, which is located in an estate owned by Mineração Joana Leite Ltda. (a bottled water company), in the Serra do Japí ridge in Jundiaí (SP), Brazil. Serra do Japí is a geological landmark, with hundreds of squared kilo- meters covered by upland type Atlantic Forest, comprising one of the largest areas of plant and animal life biodiversity in São Paulo State. Its rocks are extremely hard and are among the region''''''''s oldest (dating back more than 570 million years); they are located more than approximately 4,000 feet above sea level and were covered by glaciers in the distant past. The region has a tropical climate, with annual average temperatures of 18-20°C, and around 1,300 ml of rainfall each year.


To study the composition, safety features, in vitro efficacy and physiochemical and biological characteristics and effects of Alvorada spring mineral water.


Physiochemical features, cytotoxicity and ocular irritability
Alvorada spring mineral water''''''''s physiochemical features, cytotoxicity (viable cells) and ocular irritability (HET-CAM) were analyzed. The cytotoxicity test was carried out in cell cul- tures (fibroblasts BALB/c 3T3, clone A31). The method was based on the reduction of cellular growth and a count of viable cells, which indicates the cytotoxicity level. The irritability test (HET-CAM) was carried out with a positive control (1% SDS solution), a negative control (saline solution) and a sample of water without minerals (Milli-Q water).

Stimulation of filaggrin and aquaporin 3 genic expression
Additional studies to assess the genic expression of filaggrin and aquaporin 3 were carried out using the Real-Time PCR test. Human keratinocytes (Cascade Biologics, USA) were cul- tivated, and after six hours of contact with the studied water, the total RNA was extracted using the Trireagent ® Solution (Applied Biosystems) and quantified using the Quant-iTTM RNA Assay Kit (Invitrogen); the reading was taken with the Quibit ® Fluorometer (Invitrogen). The tests were conducted in a StepOnePlus (Applied Byosystems) device. The trials of genic expression of filaggrin were carried out using the trial system TaqMan® Gene Expression Assays (Applied Byosystems); in the analysis of the genic expression of aquaporin 3, the kit EXPRESS One-Step SYBR® GreenERTM (Invitrogen) was used. In both evaluations, the relative amount of mRNA was calculated as described by Pfaffl 1 and Gregory and Edith. 2 To evaluate the stimulation of the genic expression of filaggrin, the immunohistochemical analysis in fluorescence microscope (Leica DM 1000) of ex vivo skin fragments collected after plas- tic surgery was conducted. The antibodies used in that analysis were: anti-filaggrin primary antibody (Santa Cruz, sc-AKH1) and Alexa Flour 488 goat anti-mouse secondary antibody (Invitrogen, A11001).

Decrease in the activity of the nuclear transcription factor (NF-¿ß)
Nuclear transcription factor kappa B (NF-¿ß) activity was evaluated using a culture of human keratinocytes. The cells underwent UVA/UVB radiation and were kept in contact with the product for a further 24 hours. NF-¿ß activity was gauged using a kit marketed by Cayman Chemical, USA.

Stimulation of fibroblast proliferation
In order to evaluate the stimulation of cellular growth, fix- ation and lysis were carried out, followed by a reading of the absorbance at 260 nm. The plating was carried out in DMEM media, diluted in demineralized water (Milli-Q control), and, comparatively, in a media diluted with Alvorada spring mineral water.


Physiochemical features
Alvorada spring mineral water is characterized by the pres- ence of 15 macro and micro minerals. The Serra do Japí''''''''s min- eral water emerges at 21°C, with a 5.9 pH and no microbiolog- ic contamination, from a spring located 3,200 feet above sea level. The water''''''''s physiochemical characteristics and composi- tion are shown in Table 1.

Cytotoxicity and ocular irritability (HET-CAM)
The Alvorada spring mineral water did not present cyto- toxic activity or irritant potential in the HET-CAM test.

Stimulation of genic expression of filaggrin
The Alvorada spring mineral water''''''''s incubation in human keratinocytes culture significantly increased the relative expres- sion of filaggrin (mRNA) in the 12.5 and 6.25% (v/v) concen- trations (by 1.75 and 2.87 times, respectively), as can be verified in Graph 1. The results demonstrate a 1.5 increase in the genic expression of filaggrin (m-RNA) compared to the control group. The increased production of filaggrin was also demon- strated using the immunohistochemical technique (Figure 1) . The cuts were incubated with anti-filaggrin antibodies (in green) and DNA marker (in blue). The analyses were carried out using a fluorescence microscope.

Stimulation of genic expression of aquaporin 3
The results shown in Graph 2 demonstrate that Alvorada spring mineral water significantly increased the relative expres- sion of aquaporin 3 in the 25% and 12.5% concentrations, by 1.8 and 1.7 times, respectively.

Decrease in activity of the nuclear transcription factor (NF-¿ß)
Scientific tests with keratinocytes cultivated in vitro showed that Alvorada spring mineral water reduced the amount of NF-¿ß produced by those cells, especially after exposure to the sun (Graph 3).

Stimulation of fibroblast proliferation
The results verified that Alvorada spring mineral water stimulated fibroblast growth; there was a statistically significant difference compared to Milli-Q water, which is usually used in cell culture growth. The results are shown in Graph 4 .


The physiochemical characteristics observed show that Alvorada spring water can be classified as a mineral water, but not a thermal water, given that its emerges from the spring at 21°C – almost at room temperature. By definition, thermal water is a mineral water that emerges from its source at a min- imum of 4°C above room temperature. The verified pH value (5.9) is very close to that of the skin, indicating an excellent physiological compatibility in cutaneous conditions. The low content of dry residue (42.27 mg /L), corresponding to that of a mild water – meaning a smaller concentration of mineral salts – suggests a good acceptability in conditions that involve a com- promised cutaneous barrier. Regarding its chemical composi- tion, it is important to note the high concentration of strontium and calcium; the in vitro tests demonstrate anti-inflammatory action. In vitro tests have indicated that the other minerals pres- ent in Alvorada spring mineral water are appropriate for use in inflammatory dermatologic conditions.

Cytotoxicity and irritability (HET-CAM) tests demon- strated in vitro cutaneous and ocular safety of the Alvorada mineral water.

The genic expression tests and immunohistochemical analysis for filaggrin analyzed the mineral water''''''''s action in recovering the cutaneous barrier. The cutaneous barrier''''''''s integrity and the maintenance of the skin''''''''s hydric balance can- not only be evaluated by the presence of specific substances; the complexity of the balance among its other elements also needs to be considered. 3 Part of that complex balance is controlled by the epidermal cornified cell envelope (ECE), which is a lipopro- tein layer that substitutes the corneocytes'''''''' plasma membrane, which consists of a complex combination of interconnected proteins covalently associated to a layer with lipidic characteris- tics, attached to the extracellular surface of the protein layer. 4 Many components of the ECE, such as the pro-filaggrin, filag- grin, involucrin, loricrin, and many keratin subtypes, have been identified. 5 The epidermal protein pro-filaggrin, synthesized at a late stage during the epidermal differentiation, plays a crucial role in the generation and preservation of the flexibility and hydration of the stratum corneum (SC). 4,6 In the transition from the granular layer to the SC, pro-filaggrin (highly phosphoryl- ized) is converted into filaggrin by a specific proteolysis and dephosphorylation process. 5 The resulting filaggrin monomers combine with the intermediate keratin filaments, which are responsible for their cohesion. 7,8 In the SC, the filaggrin – a cationic protein that assists in the aggregation and subsequent disulfide cross-links among keratin filaments – is liberated from the interactions with keratin and totally degraded into its amino acid components, such as pyrrolidone carboxylic acid (PCA) and urocanic acid. 4,8,9 Those amino acids constitute around 50% of the natural moisturizing factors (NMF)s and are kept inside mature corneocytes in the SC. NMFs are crucial for maintain- ing the epidermal barrier''''''''s hydration, and are found in reduced numbers in dry or very dry skin – an effect that is intensified by the aging process and seasonal changes. In conditions that involve reduced pro-filaggrin (as in atopic dermatitis) or absent pro-filaggrin (e.g., vulgar ichthyosis), the SC''''''''s quality becomes compromised due to the deficiency of NMF and the resulting loss of transepidermal water. 10,11 The filaggrin increase that was detected by those tests can be clinically significant in the adju- vating treatment of many dermatologic disorders.

The increase in cutaneous hydration can be demonstrated by a further test that evaluates the genic expression of aquapor- in 3. Aquaporins are channels present in the plasma membranes of the cells that are responsible for the transportation of water and small solute molecules, especially glycerol, which are essen- tial for maintaining the cellular hydroelectrolytic balance of all living organisms. Aquaporins are largely distributed in cellular membranes and are part of a broader class of proteins known as integral proteins (major intrinsic protein, or MIP). Among sev- eral aquaporins, AQP3 is located in the epidermis and has a greater intensity in the basal cells'''''''' and adjacent intermediate cells'''''''' plasma membranes. 12 As epidermis cells differentiate con- tinuously, the presence of AQP3 gradually decreases until it dis- appears completely in the keratinized layer of the skin (SC). AQP3 is also present in structures associated with the epidermis, for example hair follicles and capillary glands, and acts as a spe- cialized mechanism to counterbalance the excessive loss of water. 13 Other studies reveal the capacity of aquaporin channels to carry water. In epidermal cells, the permeability to water is inhibited by mercurial agents and acidic pHs, which confirms that the transportation of water is in fact made by those chan- nels. 14 In that context, it was demonstrated that the reduction of water permeability was accompanied by changes in the perme- ability to glycerol, proving that AQP3 has an important role in the hydration of the epidermis. 15 Keratinocytes, melanocytes, fibroblasts, endothelial cells and adipocytes are equally involved in a dynamic interaction capable of detecting a variety of distur- bances in the cutaneous environment and swiftly transmitting appropriate signals that warn and recruit elements of the immunological system. 16,17 Once stimulated, those cells are capa- ble of enabling and liberating several factors that promote the expression of a great number of receptors that are significantly involved in the eicosanoids'''''''' immunoregulation and biosynthe- sis. 18,19

NF-¿ß is a protein complex that regulates immunological responses and is involved in the cellular response to stimuli such as stress and ultraviolet radiation. NF-¿ß was analyzed due to its importance in a number of dermatologic disorders. NF-¿ß is known to play a crucial role in the activation processes of genes that encode the synthesis of proinflammatory cytokines, adhe- sion molecules, chemokines, eicosanoids and nitric oxide, trig- gering a series of physiological processes that culminate in the degradation of tissue. 20,21 It is important to note that NF-¿ß is one of the main transcription factors involved in the signs and symptoms observed in the skin after acute exposure to UV radi- ation. 20 In that context, products containing active substances with anti-inflammatory and calmative properties can prevent tissular damage and photoaging caused by sun exposure.

The last in vitro test carried out analyzed the fibroblast proliferation stimulated by Alvorada spring mineral water. Cells reproduce by replicating their contents and dividing themselves in two: this cellular division cycle is the fundamental way in which all living beings reproduce themselves. In multicellular species, many cellular division cycles are required, and cellular division is necessary to substitute damaged or functionally defi- cient cells, or those that are lost through programmed cellular death (apoptosis). An adult human being needs to produce mil- lions of new cells every second to maintain this balance. If the cellular division process is damaged, for instance by an ionizing radiation dose, an individual would die in a few days. 22 The cel- lular growth index shows how long a cell takes to duplicate (or the duration of the period of cellular replication). The fibroblast proliferation observed in the study test might have happened due to the presence of the ions necessary for cellular growth – for instance, calcium ions.

All in vitro tests seem to corroborate the hypothesis that the mineral water from Serra do Japí induces s biological effects in the skin due to its physiochemical characteristics. The pres- ence of strontium in its composition makes it different from other waters available on the market. Hahn 23 demonstrated that strontium salts can be used prior to some treatments or even combined with an irritant substance, in order to inhibit sensor- ial irritation and irritative dermatitis. Zhai and colleagues tested a combination of 20% strontium nitrate and 70% glycolic acid, which also demonstrated that strontium could suppress the chemically induced irritation sensation. 24 This evidence suggests that the studied water has moisturizing properties that help rebuild the cutaneous barrier, and anti-inflammatory effects through the reduction of NF-kß activity. The role of the stron- tium also suggests that this composition can reduce skin irrita- tion. It is worth noting that the latter is involved in the phys- iopathogeny of a number of dermatologic disorders. All in vitro effects proven in this study should be tested in controlled clini- cal trials.


This study is an initial assessment of Alvorada spring min- eral water, which has shown promising results including reduc- ing inflammation, reconstructing the cutaneous barrier and restoring the skin''''''''s hydration. Its in vitro effects must be further proven in clinical trials. Since Brazil boasts one of the greatest varieties of mineral water springs, there are considerable poten- tial research opportunities relating to the different biological effects of various Brazilian mineral waters. Examining all of the physiochemical and biological characteristics of Alvorada min- eral water is very important in order to understand its clinical and dermatologic applicability.


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