*Article* **Identifying Nutrient Patterns in South African Foods to Support National Nutrition Guidelines and Policies**

**Yusentha Balakrishna 1,2,\*, Samuel Manda 2,3,4, Henry Mwambi 2 and Averalda van Graan 5,6**

**Abstract:** Food composition databases (FCDBs) provide the nutritional content of foods and are essential for developing nutrition guidance and effective intervention programs to improve nutrition of a population. In public and nutritional health research studies, FCDBs are used in the estimation of nutrient intake profiles at the population levels. However, such studies investigating nutrient co-occurrence and profile patterns within the African context are very rare. This study aimed to identify nutrient co-occurrence patterns within the South African FCDB (SAFCDB). A principal component analysis (PCA) was applied to 28 nutrients and 971 foods in the South African FCDB to determine compositionally similar food items. A second principal component analysis was applied to the food items for validation. Eight nutrient patterns (NPs) explaining 73.4% of the nutrient variation among foods were identified: (1) high magnesium and manganese; (2) high copper and vitamin B12; (3) high animal protein, niacin, and vitamin B6; (4) high fatty acids and vitamin E; (5) high calcium, phosphorous and sodium; (6) low moisture and high available carbohydrate; (7) high cholesterol and vitamin D; and (8) low zinc and high vitamin C. Similar food patterns (FPs) were identified from a PCA on food items, yielding subgroups such as dark-green, leafy vegetables and, orange-coloured fruit and vegetables. One food pattern was associated with high sodium levels and contained bread, processed meat and seafood, canned vegetables, and sauces. The data-driven nutrient and food patterns found in this study were consistent with and support the South African food-based dietary guidelines and the national salt regulations.

**Keywords:** food composition database; nutrient pattern; nutrient composition; principal component analysis; food-based dietary guideline; salt intake; South Africa


**1.2. Motivation**

48 Wireless Mesh Networks – Efficient Link Scheduling, Channel Assignment and Network Planning Strategies

Mesh routers in a WMN are stationary; therefore, the route selection should focus on discovering links that interfere with as few nodes as possible to provide high end-to-end

In contrast to traditional wireless networks, such as MANETs and WSNs, where nodes are

With the reduced cost of radios, MRs can be equipped with multiple radios. Hence, simultaneous transmission and reception can be achieved using intelligent channel

In MANETs, traffic can be from any peer Mobile Node (MN) to any other MN, while in

In MANETs, traffic distribution is generally assumed to be uniform, while in WMN, traffic

MANETs have been designed essentially for enabling communication within a small group of people, while WMNs aim to provide high-bandwidth broadband connections to a large community, and thus should be able to accommodate a large number of users accessing the Internet. Due to high estimated traffic volume in WMNs, scalability and fault tolerance

typically power-constrained, MRs have abundant power at their disposal.

**Figure 1.** A sample enterprise WMN [1]

**Mesh routers have no power constraint**

**Mesh routers have multiple radios**

**Mesh routers are static**

assignment to these radios.

**Traffic volume is high**

**The traffic model is different**

WMNs, traffic is between MRs and the GW.

**Traffic is concentrated along certain paths**

is concentrated along the paths directed towards the GW.

become important considerations in algorithm design.

throughput.

To the best of our knowledge, the proposed channel assignment algorithm is the first of its kind to use topology control based on power control for channel assignment in multi-radio multi-channel wireless mesh networks.

The main purpose of network topology control using power control is to minimize the interference between a MR and other MRs in the network by adjusting its transmission range using transmission power control. This leads to a better frequency reuse during channel assignment, which results in achieving the objective of significant improvement in the overall network throughput.


**2. Materials and Methods**

This paper primarily adopts a descriptive case study with a multiple-case design that will require investigating the phenomenon in various cases [32] such as green street manuals and constructed projects for the analysis. The primary data source for this study consists of manuals dedicated solely to green street applications; only manuals with a specific focus on such projects have been selected so that the contexts to which they refer are rigorously related to green street implementation regarding objectives and design applications. The selected manuals were primarily created by the federal, municipal agencies, or related research foundation/organizations including the corresponding codes and ordinances. Manuals were searched by such key words as green street manual and green alley manual via search engines (e.g., Google and Bing). The search engines were more efficient than using any academic databases since they were not scholarly literature but uploaded to websites or databases created by government or non-profit agencies. The primary manuals used here can be found in Table 3. In addition to the data from these manuals, data from site visits to constructed/in-progress green street projects were included, along with webpages and reports of green street cases from different cities and agencies, to ensure the breadth of the data used.

**Table 3.** *Cont.*

There were two sets of analyses: (1) A descriptive study of goals and objectives to investigate the breadth of benefits that can be generated by adopting green streets and (2) a descriptive study of green street design typologies focusing on stormwater facilities, which is one of prominent characteristics, to understand physical application of green streets within the right-of-way. An analysis based on phenomenological method suggested by Ratner [38] is conducted to identify accurate themes through multiple iteration by forming staged structures that require a rigorous analysis of the content of the data. All the contents related to objectives and benefits in the manuals were collected and "meaning units" [38], which contain specific meaning of a certain idea, were identified. Then, each identified meaning unit was summarized and grouped based on relative agendas (subcategory). The identified lists have been grouped and categorized again to get higher level criteria (top category) that can represent the lists that belong to the lower stage.

The definitions of a green street and general di fferences between green streets and typical streets were evaluated to understand the specific characteristics of green streets in the previous chapter. With the analysis, a closer look at the list of objectives in the green street manuals provided the basis for discussion about primary characteristics and the breadth of potential benefits for the surrounding communities. Typologies of green street designs applied to green street projects, especially those involving stormwater treatment facilities, were categorized according to di fferent design applications and locations where they were placed. Once the typologies were defined, a member check process was conducted with experts who worked on green street projects from governmental or non-profit agencies. The typologies were presented to the experts, whose feedback helped to modify and improve them to increase the validity of the created typologies. Overall, this paper aims to provide a comprehensive understanding of what green streets are and to work toward promoting green streets with consistent design approaches that can provide successful implementation with more benefits.


**Author details**

Fernanda Gabriela Borger1 \* and Ana Paula P. Costa2

1 FIA Business School—Administration Institute Foundation, Sao Paulo, Brazil

2 FIPE—Foundation Institute of Economic Research, Sao Paulo, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**7.1.3 Assessment of APPTRACK**

APPTRACK also has a rolling usage patterns. Again, regression analyses shows close ties between TC and SV in 2008 and 2010 and incidcates either environmental disturbances or large changes in usage patterns in 2009.

In summary, thorough analyses of a carefully designed set of relevant metrics, as well as the underlying development track history, open up many possible experiments for closely studying system dynamics over a period of time. Upon maturity, these metrics of measurements would form the foundation for developing futher statistical models to drive a set of tools for managing evolvable systems.


**14. Healthy models**

DASH and Nordic models are also healthy models, with similarities with Med Diet, and may be applied successfully, in accordance with cultural traditions and personal preferences in order to maximize adherence.

## **15. Sedentarism**

It is the fourth risk factor for death [22] and large studies are revealing a great mortality risk associated with sedentarism. Ekelund investigated in a large metaanalysis sedentary behavior effects on more than 1 million persons, revealing an association between all-cause mortality and the level of physical activity. There have been compared sitting periods of less than 4 h/day with the highest quartile of moderate or intense physical activity. One metabolic equivalent (MET) is defined as the amount of oxygen consumed while sitting at rest and is equal to 3.5 ml O2 per kg body weight × min**.** "The metabolic equivalent of task, or simply metabolic equivalent, is a physiological measure expressing the intensity of physical activities. One MET is the energy equivalent expended by an individual while seated at rest.

There is no risk for people sitting more than 8 h/day, but having more than 35, 5 MET h/week of activity (HR = 1.04, 95% CI–1.1). But those being in the lowest physical activity PA quartile, below 2.5 MET h/week and sitting <4 h/day had an increased risk. *The study conclusion is that 60–75 min/day of physical activity may attenuate or even eliminate the detrimental effect of sedentary style on health outcomes* [23, 24]. **Definition of sitting behavior** (SB): The common behavior that is considered a health threat is sitting. There are two modern definitions of SB [25].

Postural—in a sitting or reclining posture.

Contextual—walking time.

Physiological <1,5 METs.

## **16. Physical activity (PA)**

#### **16.1 Definitions (based on WHO 2020 guidelines)**

Light PA (1.6–3.0 METs), moderate (3–6 METs), and intense (>6 METs) [26]. Light-intensity physical activity.

Light-intensity physical activity is between 1.6 and 3 METs, that is, activities with energy cost less than three times the energy expenditure at rest for that person. This can include slow walking, bathing, or other incidental activities that do not result in a substantial increase in heart rate or breathing rate.

Moderate-intensity physical activity.

On an absolute scale, moderate-intensity refers to the physical activity that is performed between three and less than six times the intensity of rest. On a scale relative to an individual's personal capacity, moderate-intensity physical activity is usually a 5 or 6 on a scale of 0–10; intense PA is at a level higher than 6 MET's..

#### **16.2 Physical activity**

This is essential for health and is an important component of a healthy lifestyle. Promoting continuously all PA benefits will lead finally to a higher percentage of people adopting healthy behaviors. The latest WHO guidelines [26] include the major developments vs. 2010 guidelines, being realized based on larger scientific evidence. Additional health benefits are supported by studies-cognitive health improvement, mental health, sleep, and health-related quality of life, and are emphasized beyond traditionally known benefits for cancer prevention, metabolic diseases prevention, cardiorespiratory fitness improvement, and musculoskeletal and functional health. All these documents are reflecting a maturity of the research, but also the complexity of WHO's definition of health as "a state of complete physical, mental and social wellbeing" [27].

#### *Lifestyle Factors and Obesity DOI: http://dx.doi.org/10.5772/intechopen.100254*

Mentioning that a relation between cardiovascular cause mortality, all-cause mortality, and PA is well proved, WHO guidelines are reaffirming that any level and all intensities of PA are associated with low mortality risk. The incidence of type 2 diabetes is decreasing proportionally with PA level.  Meanwhile, adiposity is inversely related to PA and sleep and quality of life (QOL) may be considerably improved according to the level of PA. Development of depression and anxiety may be slower for active people. Guideline's conclusions are that any level of any intensity of PA is associated with lower mortality from all causes but also with reduced incidence for type 2 diabetes, hypertension, and cardiovascular disease.

## **16.3 Recommendations for adults 18: 64 years**

There are specific recommendations for limiting sedentary behavior, which are as follows:

### **16.4 Recommendations for older adults**

Older adults (> 64y) usually have a very low level of physical activity. WHO guidelines are emphasizing rules even for this period of life, bringing the same benefits as for the other adults. Additionally, for older adults, PA may prevent falls and injuries, a decline in bone density and functionality also will attenuate the decline of muscular mass.

Recommendations for older adults are (as described by WHO guidelines 2020) as follows:

• PA should be regularly performed by any older adults.

Important for older adults is that any activity is better than inactivity and PA should be increased gradually, based on personal functional capacities and fitness level.


The threats that affect the mangrove are several; among which we highlight tourism, industries, infrastructure and deforestation. The methodology used to find out the conservation status of these ecosystems is based on the phytosociological method. With this method, 16 plant communities have been described, which present ecological and floristic differences. Each plant association presents its own characteristic species (Acar), and companion species (Acom) belonging to neighbouring communities. For this reason, and for the first time, we take stock of the relationship between characteristic and companion species, and in response to this, the state of conservation of the plant association. The state of conservation of the mangrove is high when all its species are characteristic, as this ecosystem is poor in characteristic species, its conservation is good, but if it presents a high diversity, it means that it presents many opportunistic companion species, and the

The floristic diversity presented by some mangrove communities is not synonymous with a good state of conservation, but rather the reverse: this diversity is a cause for concern, as it is due to the high number of invasive species that are difficult to eradicate while the current threats are maintained, in the form of cutting, burning, forest fires, charcoal manufacture, and

Therefore, the best conserved mangrove communities are those which present only typical mangrove species and no companions, even in the case of monospecific populations of *Rhizophora mangle, Laguncularia racemosa, Avicennia germinans, Conocarpu erectus.* The mangrove forest must be regarded as a fragile ecosystem as it demands ecological conditions of depth of water, salinity, and a very specific substrate, and in which any alteration triggers the

Based on the results obtained, we propose concrete measures to mitigate and prevent the

**1.** Not to carry out deforestation in peripheral areas to avoid erosive phenomena and the

deviation and substitution of these communities by neighbouring ones.

**2.** Deforestation with the aim of obtaining energy (coal) must be prohibited.

**3.** Implement policies for the integration of rural populations in their environment.

a significant degree of threat.

204 Mangrove Ecosystem Ecology and Function

state of conservation the mangrove is bad.

destruction of the mangrove communities:

consequent filling of the lagoon vessel.

**4.** Control of mass tourism.

**4. Conclusions**

so on.

We would like to thank Ms. Pru Brooke Turner (MA Cantab.) for the English translation of this article, and the architect Francisco Javier Quiros Higueras for developing the vegetation profiles.

### **Author details**

Ana Cano-Ortiz1 , Carmelo Maria Musarella1,2, José Carlos Piñar Fuentes1 , Carlos Jose Pinto Gomes3 , Sara Del Rio4 , Ricardo Quinto Canas5 and Eusebio Cano1 \*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Animal and Plant Biology and Ecology, Botany Section, University of Jaén, Jaén, Spain

2 Department of AGRARIA, "Mediterranea" University of Reggio Calabria, Reggio Calabria, Italy

3 Department of Landscape, Environment and Planning/Institute of Agricultural and Environmental Sciences, Mediterranean (ICAAM), University of Évora, Evora, Portugal

4 Department of Biodiversity and Environmental Management (Botany), Faculty of Biological and Environmental Sciences, University of León, León, Spain

5 CCMAR – Centro de Ciências do Mar, Universidade do Algarve, Faro, Portugal

### **References**

[6] Reyes OJ, Acosta Cantillo F. Fitocenosis presentes las áreas costeras del sur de la Sierra Maestra, Cuba. I. Comunidades con influencia marina. Foresta Veracruzana.

[7] Van der Maarel E. Transformation of cover-abundance values in phytosociology and its

[8] Cano E, García A, Nieto J, Torres JA. Estudio de la evaluación de hábitats de Laguna Honda (Jaén, España). Departamento de Ecología. Universidade de Évora, Actas: I colo-

[9] Mendes LW, Tsai SM. Variations of bacterial community and composition in mangrove

[10] Cano E, Veloz Ramirez A, Cano Ortiz A, Esteban FJ. Analysis of the *Pterocarpus officinalis* forests in the gran Estero (Dominican Republic). Acta Bot. Gallica. 2009;**156**(4):559-570

[11] Cano Ortiz A, Musarella CM, Piñar JC, Veloz A, Cano E. The dry forest in the Dominican

[12] Lugo AE, Snedaker SC. The ecology of mangroves. Annual Review of Ecology and

[13] Joshi HG, Ghose M. Community structure, species diversity, and aboveground biomass

of the Sunderban mangrove swamps. Tropical Ecology. 2014;**55**(3):283-303

sediment at different depths in southeastern Brazil. Diversity. 2014;**6**:825-843

effects on community similarity. Vegetatio. 1979;**39**:97-114

quio internacional de ecología da vegetaçao; 1996. pp. 265-275

Republic. Plant Biosystems. 2015;**149**(3):451-472

Systematics. 2003;**5**:39-64

2003;**5**(002):1-8

206 Mangrove Ecosystem Ecology and Function

### *Edited by Sahadev Sharma*

*Mangrove Ecosystem Ecology and Function* deals with several aspects of mangrove science, as well as conservation, management, and related policies. The book is divided into six sections and structured into 10 chapters. The first section discusses mangrove ecology, structure, and function; the second section explains mangrove physiology related to salt accumulation; the third section focuses on mangrove polychaetes; the fourth section talks about the bioprospect of mangrove microbes; the fifth section discusses soil geochemistry; and the sixth section elucidates mangrove management and conservation. Researchers from different countries and fields of mangrove ecosystem exploration have contributed their findings. This book would be an ideal source of scientific information to graduate students, advanced students, researchers, scientists, and stakeholders involved in mangrove ecosystem research.

Published in London, UK © 2018 IntechOpen © atosan / iStock

Mangrove Ecosystem Ecology and Function

Mangrove Ecosystem

Ecology and Function

*Edited by Sahadev Sharma*


**3. Results**

#### *3.1. ISL Suppressed Breast Cancer MDA-MB-231 Cell Growth*

The MTT assay was performed to examine whether ISL can suppress breast cancer MDA-MB-231 cell growth. The results showed that cell viability was reduced by treatment of cells with ISL at 25 and 50 μM for 48 and 72 h (Figure 1a), whereas treatment of cells with ISL at 25 and 50 μM for 48 and 72 h was shown to increase cytotoxicity by LDH activity analysis (Figure 1b). The cell morphology and number were also monitored, and we found that the cell morphology switched from the spindle type to irregular by treatment of cells with ISL at 25 and 50 μM for 48 h (Figure 1c). Furthermore, a reduction of the cell number was observed by treatment of cells with 25 and 50 μM for 48 h (Figure 1d).

**Figure 1.** Inhibitory e ffects of isoliquiritigenin (ISL) on the proliferation of breast cancer MDA-MB-231 cells. (**a**) MDA-MB-231 cells were seeded in 96-well plates (3000 cells per well) with 100 μL per well culture medium. Cells were treated with ISL in various doses for 24, 48, and 72 h. At the end of incubation, cell viability was measured by MTT assay (*n* = 3). (**b**) MDA-MB-231 cells (2 × 104 cells per well) seeded in 96-well plates. Cells were treated with various doses for 24, 48, and 72 h. The cytotoxic effects of ISL was detected using the lactate dehydrogenase (LDH) assay kit (*n* = 3). (**c**,**d**) MDA-MB-231 cells (2 × 105 cells per well) were seeded in six-well plates. After treatment of cells with ISL for 48 h, the cell number was monitored (*n* = 4). Data are represented as mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group.

#### *3.2. ISL Suppressed the Cell Cycle Progression of MDA-MB-231 Cells*

Here, we studied whether ISL suppressed cell growth by inflecting the cell cycle progression. MDA-MB-231 cells were treated with ISL and then the cell cycle distribution was monitored using flow cytometry analysis. The results showed that treatment with ISL at 25 and 50 μM for 48 h reduced the cell population in the G1 phase in MDA-MB-231 cells (Figure 2a,b). In addition, the expression of G1/S gate-associated proteins CDK4 and cyclin D was detected. The results showed that treatment with ISL did not alter the expression of CDK4 (Figure 2c), but the expression of cyclin D1 was reduced by treatment with ISL at 25 and 50 μM for 48 h (Figure 2d).

**Figure 2.** Cell cycle progression of MDA-MB-231 cells was suppressed by treatment with ISL. (**a**) MDA-MB-231 cells were treated with ISL for 48 h. Cells were stained with propidium iodide, and the cell cycle distributions were analyzed by the flow cytometry. (**b**) The quantitative results of cell distributions in MDA-MB-231 cells are shown. Cell-cycle-associated proteins (**c**) CDK4 and (**d**) CyclinD1 were analyzed using Western blot. Each target protein was normalized to GAPDH expression. Data are represented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group.

#### *3.3. ISL Induced Apoptotic Cell Death*

To further examine whether ISL induced cell apoptosis in MDA-MB-231 cells, the cells were stained with annexin V FITC, and then the apoptotic cell population was analyzed by flow cytometry. The results showed that the annexin-V-positive (early apoptotic phase) and annexin-plus-PI-positive (later apoptotic phase) cell populations were increased by ISL treatment at 25 and 50 μM for 48 h (Figure 3a,b). The expression of Bcl-2 protein decreased from ISL treatment at 50 μM for 48 h (Figure 3c,d), whereas treatment with ISL increased the expression of Bax protein (Figure 3c,e). In addition, the cleaved form of caspase-3 was induced by treatment with ISL at 25 and 50 μM for 48 h (Figure 3f,g), and the expression of cleaved PARP was also increased by treatment with ISL at 50 μM for 48 h (Figure 3f,h).

**Figure 3.** ISL induced cell apoptosis by upregulating apoptotic protein expression in MDA-MB-231 cells. (**a**) MDA-MB-231 cells were treated with ISL for 48 h. Cells were stained with propidium iodide and annexin V fluorescein isothiocyanate (FITC), and the apoptosis rates were analyzed by flow cytometry. (**b**) The quantitative data of apoptotic cell death in early and late phases are shown. (**c**) After treatment as indicated above, the anti- and proapoptotic proteins were monitored using Western blotting in MDA-MB-231 cells. (**d**,**e**) Each target protein was normalized to GAPDH or β-actin expression. (**f**) The expression of caspase-3 and its downstream molecule, PAPR, was monitored using Western blotting in MDA-MB-231 cells. (**g**,**h**) Each target protein was normalized to β-actin expression.

Data are represented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group. + *p* < 0.05 compared with the early apoptotic phase of the control group. # *p* < 0.05, ## *p* < 0.01 compared with the later apoptotic phase of the control group.

#### *3.4. ISL-Mediated p62 Accumulation Causes Autophagy-Mediated Apoptosis*

The total and phosphorylated protein levels of mTOR decreased after treatment with ISL at 50 μM for 48 h (Figure 4a,b). The downstream molecule ULK1 was activated by mTOR regulation in the autophagy pathway [25]. We found that the total and phosphorylated protein levels of ULK1 decreased after ISL treatment at 10, 25, and 50 μM for 48 h (Figure 4a,c). The autophagosome-associated proteins p62, Beclin1, and LC3 were increased by treatment with ISL for 48 h (Figure 4d–f). In our results observing that p62 accumulation, with autophagy inhibitor bafilomycin (BAF), blocked p62 accumulation and caused cell toxicity (Supplementary Figure S1). In the autophagy process, lysosomal proteases are needed, such as Cathepsins degrades p62 [26]. Treatment with ISL at 50 μM for 48 h significant decreased cathepsin B protein expression (Figure 4g), which explains the p62 accumulation. Moreover, p62 accumulation was related to the increased apoptosis through the increase of caspase-8. Furthermore, we used siRNA to eliminate p62 function. After 48 h, ISL increased caspase-8 protein expression. However, ISL combined with siRNA treatment have a lower expression of caspase-8 than ISL treatment (Figure 4h).

**Figure 4.** ISL treatment induced the expression of autophagy-associated proteins in MDA-MB-231 cells.

MDA-MB-231 cells were treated with ISL for 48 h. (**a**) The expression levels of mTOR and ULK1 protein were analyzed by Western blotting. The total and phosphorylated forms of (**b**) mTOR and (**c**) ULK1 were normalized to β-actin expression. The expression levels of (**d**) p62, (**e**) Beclin1, (**f**) LC3, and (**g**) Cathepsin B were also analyzed using Western blotting. MDA-MB-231 cells were used lipofectamine 3000 (Thermo Fisher Scientific) to transfect SignalSilence® SQSTM1/p62 (Cell Signaling), and treated with ISL for 48 h. The expression of (**h**) caspase-8 proteins were also analyzed using Western blotting. Data are represented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group. # *p* < 0.05, ## *p* < 0.01 compared with the phosphorylated protein level of the control group.

#### *3.5. Preventive E*ff*ects of ISL on Breast Cancer Cell Growth in a Xenograft Mouse Model*

A breast cancer MDA-MB-231 cell xenograft mouse model was generated to evaluate whether ISL has a potential role in antitumor proliferation in vivo. After pretreatment with PBS or ISL at 2.5 and 5.0 mg/kg for 2 weeks, MDA-MB-231 cells were inoculated into mice, which were constitutively treated with ISL for an additional 25 days. The results showed that MDA-MB 231 tumors formed in all five mice by treatment with PBS. However, the number of MDA-MB 231 tumors was reduced by pretreatment with ISL at 2.5 mg/kg (three of five mice) and 5.0 mg/kg (two of five mice) (Figure 5a,c). Measuring the tumor volume elicited by MDAMB-231 cells showed that the tumor volume increased by treatment with PBS in a time-dependent manner, and ISL treatments significantly reduced them (Figure 5b). At the end of the experiment, tumor tissues were isolated and weighed. Treatments of mice with ISL at 2.5 and 5.0 mg/kg showed significantly reduced tumor weight compared with the vehicle group (Figure 5d). According to the IHC results, expression of the Ki-67 protein level was observed in tumor tissues for mice treated with ISL at 2.5 and 5.0 mg/kg, separately (Figure 6, top panel). Treatment with ISL at 5.0 mg/kg increased the caspase-3 expression level in tumor tissues (Figure 6, middle panel). Moreover, the expression of p62 was increased in tumor tissues by treatment with ISL at 2.5 and 5.0 mg/kg, separately (Figure 6, bottom panel).

**Figure 5.** ISL suppressed tumor growth, as shown by the human MDA-MB-231 breast tumor xenograft

mouse model. Mice were pretreated with ISL at 2.5 and 5.0 mg/kg or PBS as control by oral gavage once daily for 2 weeks. Then, MDA-MB-231 tumor cells (5 × 106 cells per mouse) were implanted into mice by subcutaneous injection on the right flank and the mice received oral gavage of PBS and/or ISL for 25 days. At the end of the experiment, (**a**) the mice were photographed, (**b**) tumor size was monitored, (**c**) tumors were isolated and photographed, and (**d**) tumor weight was recorded. Data are represented as mean ± SEM (*n* = 5). \*\* *p* < 0.01 compared with the control group.

**Figure 6.** Representative results of IHC staining for Ki-67, caspase-3, and p62 protein expression levels in ISL-treated MDA-MB-231 breast tumor xenograft mouse model. MDA-MB-231 tumor cells (5 × 106 cells per mouse) were implanted and mice were treated with ISL for 25 days Then, tumor tissues were isolated and immunohistochemistry was performed to analyze Ki-67, caspase-3, and p62 protein levels. Images were photographed (200× magnification; scale bar = 200 μm).

#### *3.6. ISL Suppressed VEGF Production and Capillary-Like Tube Formation of SVEC4-10 Cells*

IHC analysis was performed to evaluate the expression of VEGF in tumor tissues by treatment with ISL. The results showed that the expression of VEGF was significantly reduced in treatment of mice with ISL at 2.5 and 5.0 mg/kg, separately (Supplementary Figure S2a). Measuring the serum VEGF level showed that treatment with ISL at 2.5 and 5.0 mg/kg reduced the serum VEGF level compared with the vehicle group (Supplementary Figure S2b). Additionally, to further investigate whether ISL has a potential role in antiangiogenesis, we performed a capillary-like tube formation assay. The results showed that the capability of capillary-like tube formation in SVEC4-10 cells was inhibited by treatment with ISL at 10 μM for 5 h (Supplementary Figure S3).


**4. How does trauma change your health?**

Traumatic events are now known to change not only psychological well-being but also the basic structure of how the body perceives painful events, processes them and produces physical reactions. But how does this happen? How do traumatic events in childhood such as sexual abuse change health status? It is important to note that not all individuals exposed to traumatic events will experience noxious outcomes, therefore genetic predisposition/vulnerability (Neigh, Gillespie, Nemeroff, 2009) and environmental factors are suspected when examining etiology. Stern's oft quoted metaphor (Kazaks & Stern, 2005) "Genetics load the gun, and environment pulls the trigger," appears apropos.

Humans have the capacity to react to stressful events in ways that will protect them. Many people have heard of the fight/flight/freeze reaction to threatening events. This is the body's stress reaction alerting the individual to either stay and fight, run away from the stressful event, or sometimes freeze in place. Each reaction is designed to assist the individual to survive and is directly attributable to the autonomic nervous system and the endocrine system working together to deal with the stressful event.

one cannot escape. CSA survivors with chronic PTSD often experience hyperarousal and hypervigilience that are continuous physiological states. Chronic PTSD frequently includes the inability to self-soothe and emotionally regulate. Health risk behaviors such as alcohol and substance abuse, cigarette smoking, eating disorders, and self-injurious behavior (cutting) are correlated with childhood sexual abuse. These health risk behaviors can be considered attempts by the CSA survivor, albeit poor ones, in the adaptive struggle in which self-medicating is necessary for survival.  Because the nature of sexual abuse is chronic, usually perpetrated by a caregiver, and occurs during childhood, it impacts critical periods of neurobiological development (van der Kolk, 2011). When caregivers are abusive, absent or neglectful, and/or helpless in the face of trauma, children cannot develop a sense of safety and stability that emanates from the caregiver nor can they rely on that caregiver to restore a sense of calm and reliability. The child's responses to stress become diffuse and inadequate. The ability to self-soothe and emotionally regulate – managing stress, impulsivity, and anxiety – are markedly impaired. A significant issue is that the child experiences a sense of betrayal

Mental health practitioners and researchers have examined the concept of complex PTSD (Herman, 1992a, 1992b) and the tentative diagnosis of Developmental Trauma Disorder (Cook et al, 2005; van der Kolk, 2005). Cook et al (2005) state, "A comprehensive review of the literature on complex trauma suggest seven primary domains of impairment observed in (traumatically) exposed children: attachment, biology, affect regulation, dissociation, (e.g., alterations in consciousness), behavioral regulation, cognition, and self-concept" (p. 392). Included in these domains are the child's worldview and functional impairments such as academic, interpersonal, legal interactions, and vocation (van der Kolk, 2005). This tentative diagnosis, yet to be wholly accepted by the trauma field, appears to encompass the chronic

Traumatic events are now known to change not only psychological well-being but also the basic structure of how the body perceives painful events, processes them and produces physical reactions. But how does this happen? How do traumatic events in childhood such as sexual abuse change health status? It is important to note that not all individuals exposed to traumatic events will experience noxious outcomes, therefore genetic predisposition/vulnerability (Neigh, Gillespie, Nemeroff, 2009) and environmental factors are suspected when examining etiology. Stern's oft quoted metaphor (Kazaks & Stern, 2005)

Humans have the capacity to react to stressful events in ways that will protect them. Many people have heard of the fight/flight/freeze reaction to threatening events. This is the body's stress reaction alerting the individual to either stay and fight, run away from the stressful event, or sometimes freeze in place. Each reaction is designed to assist the individual to survive and is directly attributable to the autonomic nervous system and the

"Genetics load the gun, and environment pulls the trigger," appears apropos.

endocrine system working together to deal with the stressful event.

and perceives the world as a hostile and attacking environment.

and devastating nature of trauma to the developing child.

**4. How does trauma change your health?**

memories.

The hypothalamic-pituitary-adrenal (HPA) axis is a mediating pathway of the stress response (Neigh, Gillespie, Nemeroff, 2009, van der Kolk, 2011) and it's the function of the HPA to modulate hormones that address stressful events. To promote survival, a chain reaction of powerful hormones and neurochemicals are produced to assist the individual in dealing with the immediate stressful event (Neigh, Gillespie, Nemeroff, 2009). Deactivation of these hormones occurs through a "negative feedback loop" alerting the individual that danger is no longer present. Neigh, Gillespie, and Nemeroff (2009) state,

However, if the stress response becomes chronic due to repeated exposure to stressors, defects at different levels of the negative feedback system, or both, the result is a sustained increase in the level of stress hormones and the initiation of pathological changes across multiple physiological systems, resulting in stress related diseases (p. 391; also see McEwen, 2008).

The sympathetic nervous system (SNS) is dominant over the parasympathetic nervous system (PNS), and will not yield to the PNS until some form of resolution takes place. Resolution can take the form of fighting, jogging, meditating (Howard, 2006). The adrenal glands release enough adrenaline to get your attention at the first sign of stress. Adrenaline also helps to "imprint" an emotional or traumatizing event. If the stress continues, the hypothalamus secretes corticotropin-releasing factor. In this process cortisol is also released.

Cortisol is a steroid hormone that is produced naturally in the body to assist in the adaptive struggle when an individual is facing acute traumatic situations. When the stress or the traumatic situation is chronic, high levels of cortisol become toxic. Prolonged cortisol production impairs the immune system and thus, healing. Vulnerability to stress-related disorders and diseases, such as gastrointestinal disorders (ulcers) and heart disease commences. Chronic trauma and stress induces lower cortisol production and this decrease creates an enhanced autoimmune system. In the absence of any other illnesses, the autoimmune system will attack various systems within the body and create illnesses such as Fibromyalgia, Chronic Fatigue Syndrome, thyroid diseases, and Krohn's disease (Bergmann, 2011). Howard (2006) states,

Minor results of this stress-related impairment include colds, flu, backaches, tight chest, migraine headaches, tension headaches, allergy outbreaks, and skin ailments. More chronic and life-threatening results can include hypertension, ulcers, accidentproneness, addictions, asthma, infertility, colon or bowel disorders, diabetes, kidney disease, rheumatoid arthritis, and mental illness. Killers that can result include heart disease, stroke, cancer, and suicide (p. 816).

Chronic trauma, coupled with the severity of the abuse itself, has long-term devastating impact on health and mental health due to the chronicity of these physiological states.

### **5. Mental health features of the CSA survivor that impede their health outcomes**

CSA survivors may experience shame and self-blame regarding the sexual abuse and approach both mental health practitioners and health professionals with distrust, fear, and anxiety. CSA survivors may also experience a range of PTSD symptomatology that includes avoidant behaviors, depression, and dissociation.

Childhood Sexual Abuse and Adult Physical and Dental Health Outcomes 143

2011). All of these factors modulate functional aspects of the gastrointestinal system and later, symptoms. While IBS is influenced by several factors such as genetics, environment,

Gynecological problems such as difficult menses and pain when having sexual intercourse, sexual performance, as well as promiscuity, may also contribute to poor reproductive health

CSA survivors may also suffer from Immune System Dysfunction, musculoskeletal difficulties, respiratory ailments such as Asthma, and rheumatic disorders. CSA survivors can also experience what is called Medically Unexplained Symptoms (MUS) such as fibromyalgia (Roelofs & Spinhoeven (2009). Other types of difficulties include urinary tract infections, migraine headaches, chronic pelvic pain and pain intolerance or sensitivity.

Addictive behaviors that lead to poor health outcomes are also associated with childhood sexual abuse such as eating disorders (anorexia, bulimia, and obesity), substance abuse (alcohol and/or drugs), and cigarette smoking. Table 1 lists the most common medical

Dental health has gained increasing attention as a primary factor supporting overall health. Periodontal disease is now associated with upper respiratory illnesses such as pneumonia and cardiac conditions, even premature death (Eke, Thornton-Evans, Wei, Borgnakke, Dye, 2010). Given the importance of dental health in overall well-being, it is significant that by and large CSA Survivors have poor dental health, health seeking behaviors, and follow through with dental protocols (Hays & Stanley, 1996; Leeners, Stiller, Block, Gorres, Imthurn, Rath, 2009; Monahan & Forgash, 2000; Willumsen, 2004). As with physical health, dental health seeking behaviors can be defined as recognizing the necessity for dental care,

and family environment, childhood sexual abuse is a contributing factor.

outcomes.

conditions for CSA survivors.

 Autoimmune Diseases Cardiovascular Disease

Gynecological Issues

Rheumatic Disorders

Diabetes

 Pain Perception and Chronic Pain Compromised Reproductive Health

 Immune System Dysfunction Musculoskeletal difficulties Respiratory Ailments (Asthma)

Eating Disorders (Anorexia, Obesity, Bulimia)

**9. Dental health issues for CSA survivors**

Gastrointestinal Disorders (Irritable Bowel Syndrome)

Medically Unexplained Symptoms (MUS) e.g., fibromyalgia

Table 1. Common Medical Difficulties Reported By CSA Survivors

The survivor may not have had good health care models to emulate or his or her health care needs may have been neglected and minimized. Cycles of negative emotions, stress, pain and self-defeating beliefs may perpetuate physical problems. Years of avoidant behavior regarding health issues may have passed before the CSA survivor reaches the health care professional, thus warranting more intensive and costly treatment.

### **6. Health risk behaviors**

In an effort to self-soothe, the CSA survivor may have tried to self-medicate using a variety of maladaptive behaviors and methods. No one would doubt that the prevalence of addiction difficulties with substances such as drugs and alcohol in the United States is of epidemic proportion.   Self-injurious behavior such as skin carving has long been thought to alleviate some of the internal stress experienced by CSA survivors.

### **7. Medical issues**

Before reviewing the medical issues of CSA survivors one must consider the often mistaken assumption that the somatic complaints of the survivor have no physical etiology, and therefore they are often overlooked and/or misdiagnosed. This phenomenon occurs both with the health and mental health care professional as well as the CSA survivor.

Psycho-physiological changes coupled with psychological changes create multi-systemic problems common to CSA survivors. The American Medical Association 1992 addresses these changes when they state,

The event has such physiological and psychological intensity that it overrides and impairs the individual's neurophysiological mechanisms of adaptation. The resulting damage is not merely emotional. The person's biological capacity to tolerate and regulate internal and external stimulation can be altered. These changes, in turn, compromise the person's ability to organize perceptual stimuli and cognitive information, making them susceptible to a range of somatic illnesses and a spectrum of anxiety and depressive disorders (p 35).

Psychological distress also contributes to the CSA survivor having an impaired view of their health either through somatization and/or dissociation.