**1. Introduction**

Caffeine (1,3,7-trimethylxanthine) is the substance most frequently consumed in our daily life. For example, coffee, most of the soft drinks and over-the-counter medications contain caffeine [1]. On the other hand, cocoa contains theobromine (3,7-dimethylxanthine). The main source of theobromine which is daily consumed by humans from childhood to adult is chocolate. Cocoa produces no adverse effect, in normal dosage [2]. These two similar families of xanthine, but the opposite properties on the crystal formation of the hydroxylapatite (HAP) of the teeth were discovered accidentally. Fluoride is the only known chemical that affected the HAP of the enamel by converting it to fluorohydroxylapatite.

In the dental community, fluoride has been used not only as the main ingredient of dentifrice but is also used in many others. However, recently fluoride was designated as one of the developmental neurotoxicants as more adverse effects of fluoride are revealed [3]. Although besides the developmental neurotoxicants, fluoride is also known to affect the pineal gland [4] and thyroid gland [5] and there is even some evidence between bone disease and fluoride exposure [6].

In addition, there are many reports [7–12] that infants, newborns and young children were exposed to fluoride and fluorosis among them is common. Unfortunately, in dentistry, fluorosis was mainly considered as an esthetic problem to which no more serious consideration is paid attention. Recently, it was proposed that fluoride exposure in early life may become a root cause of the disease in later life [13].

Pups' teeth were affected by the sucking milk of lactating dams which were given a caffeine-containing diet [14]. (In each animal experiment described, we obtained the permission to use the animals from the animal care committee at the LSU Health Sciences Center.) Then, eventually, non-fluoride dentifrices were developed.

### **2. The effect of caffeine on enamel**

#### **2.1 Dissolution studies**

Further studies were conducted to determine whether the effects of caffeine come from either enamel or dentin, or both. If certain effects come from enamel, it would be an extremely interesting phenomenon, because the only chemical known to affect the HAP of the enamel is fluoride. In addition, any changes in the HAP on the enamel by caffeine might be linked to possible incidences of future dental caries.

Using the method described [15] on how to study the enamel surface of the teeth which were affected by the nutritional deficiency, the condition of the enamel was studied. The samples from the first or second molars were obtained at postnatal day 22 which is the end of lactation. The teeth were exposed to weak acid for 80 minutes and four fractions were collected at 20 minutes intervals. The experimental procedures were described in detail [16, 17]. The enamel surface of secondary electron photomicrographs are shown in the control (**Figure 1**) and caffeine group (**Figure 2**).

The apparent effects of crystallization by caffeine on the enamel of first molars where calcium, phosphorus and magnesium were released more of the teeth in the caffeine exposed offspring (**Table 1**). The first molars showed a statistically significant amount of dissolution in the caffeine group of the respective ions measured for 80 minutes compared to the non-caffeine control group [16].

On the other hand, there is no significant difference between caffeine and the control group in each mineral dissolved in the second molars.

This difference in the caffeine's effects between first and second molars can be explained as follows. The first stage of development is called the hyperplastic growth period which is primarily an increase of DNA of the organ. Synthesis of DNA and cell division at first take place rapidly [18], but thereafter, slow down. Further tissue growth can occur by cytoplasmic enlargement.

*The Contrasting Effects between Caffeine and Theobromine on Crystallization… DOI: http://dx.doi.org/10.5772/intechopen.101116*

#### **Figure 1.**

*Left: Before acid exposure in the control. Right: 80 minutes after the acid exposure.*

#### **Figure 2.**

*Left: Before acid exposure of the caffeine group. Right: 80 minutes after the acid exposure.*

The measured DNA content as an index of cell number distinguishes growth by an increase of cell number (hyperplasia). Cell division eventually ceases will be seen to determine when the organ is no longer vulnerable to nutritional stress. If nutritional stress, such as caffeine exposure in the early part of life were applied hyperplastic growth period, the organ or body would never recover to the original condition. Therefore, this period is called a critical growth period.

The second stage is a gradual decrease in cell number and a slow increase of cell size in the organ. The third stage is primarily an increase in cell size, which is called the hypertrophic growth period. If the nutritional stress were applied in this period, organ or animal grows back normally, provided that enough nutrition is given [19, 20].

During the period of growth and development, the critical period of growth is the most important concept. For example, the huge increase of DNA in the brain occurs primarily during gestation and early lactation period whereas an increase of the DNA of the heart continues until adulthood [21]. This indicates that the critical period of growth is different among the organs, therefore, the effects of the nutritional stress on each organ is different, depending upon when the stress is applied.

Likewise, caffeine exposures for the growing period were different between the first and second molars, indicating that the critical period of growth during caffeine exposure was different between the first and second molars. First molars are affected


*Each value is an average of seven determinations.\* Significantly different from the control at P < 0.05.*

#### **Table 1.**

*Mean amount of minerals dissolved from first or second molars during each time interval (*μ*g/four first or second molars; mean ± SEM).*

by caffeine intake by the offspring during this period. Therefore, the experiments were conducted only using the first molars.

#### **2.2 Crystallization studies of HAP**

To find out what is happening to the enamel of the first molars, samples that were not used in the studies were powered and enamel was separated from dentin [22]. Then a pure enamel sample was run for 4 hours on a Gandolfi X-ray powder camera. Also, various other aspects of the samples were studied in detail [17].

The filmstrip run on the Gandolfi camera recorded more diffuse lines for the samples of the caffeine group compared to the control (**Figure 3**).

X-ray diffraction analysis on enamel samples by the Gandolfi X-ray camera showed the caffeine supplementation in the maternal diet affected mineralization of enamel, as broader lines indicated smaller crystallites of enamel. Smaller crystallites increase susceptibility to dissolution.

This explains why the caffeine group of the teeth showed the higher dissolution of each ion from the surface of enamel throughout the experimental period [16].

#### **2.3 Cariogenic studies**

Because the teeth were affected by caffeine it is natural to make a simple assumption, that is, is it possible to produce in vivo dental caries in the caffeine group [16].

*The Contrasting Effects between Caffeine and Theobromine on Crystallization… DOI: http://dx.doi.org/10.5772/intechopen.101116*

**Figure 3.** *Top: control. Bottom: caffeine group.*

Thus, the experiment was conducted by raising the offspring the same way as above. At weanling on postnatal day 22, offspring were fed the cariogenic diet until day 50 (a total of 28 days) to see whether the caffeine group show a response in dental caries using the methods described [23]. The caffeine group showed significantly higher caries scores than that of the non-caffeine control (*P* < 0.05) (3.36 ± 0.33 versus 2.65 ± 0.22) (mean ± SEM) [24].

Therefore, the hypothesis turned to be true as is shown in **Figures 4** and **5**. The amount of caffeine that was added to the maternal diet was 2 mg/100 g bodyweight of the dam. The equivalent comparison between the caffeine in the rat and human is based on metabolic body weight (kg0.75) [25]. (Metabolic rates are expressed in terms of metabolic body size—i.e., kg0.75, the point at which the dependence on different body sizes disappears.) The human caffeine intake is comparable with slightly more than two cups of coffee daily.

This is the normal amount of caffeine consumed by humans. Although extrapolation from rat data to human dental caries incidence requires extreme caution, nevertheless present data indicate that caffeine exposure during the early growth period impairs the structure of the enamel crystal formation of the developing teeth. Particularly, it may require attention where the offspring born from a pregnant woman who habitually consumes caffeine-containing soft drinks and/or coffee drinkers may develop teeth that are prone to dental caries of the offspring in the future. It seems clear that caffeine exposure during the critical growth period affects the amelogenesis of the enamel, affecting the crystal size of the HAP during mineralization.

#### **Figure 4.**

*Molar of the control at day 50 stained with 0.06% murexide in 70% ethanol for 16 hours (top), the caffeine group (bottom).*

**Figure 5.** *Secondary electron image of control at day 50 (left) and the caffeine group (right).*
