*3.1. Biological Factors*

Five participants reported easy adjustment to a 16-h fast, agreeing that fasting got easier as the study went on. Seven participants reported that it was not difficult to eat enough calories within the 8-h window. After the first few days of fasting, only one participant reported uncomfortable hunger during the study, but this participant misunderstood the protocol and was only eating one meal a day. Six of the nine participants stated that overeating was not problematic during the eating window and two participants neither agreed nor disagreed. Normal energy levels were retained throughout the intervention, with eight of the nine participants reporting "disagree" when asked if energy levels decreased while fasting. Only one participant indicated that fasting interfered with normal sleep patterns. Participant responses to the five questions within the biological domain of the Diet Satisfaction Survey are displayed in Figure S3.

## *3.2. Psychological Factors*

Subjective mood and quality of life were unaffected in six of the nine participants, and seven participants expressed eagerness to participate in a similar study again. Despite this, participant comprehension of the TRF protocol was lower than expected, with many participants not fully understanding instructions regarding calorie consumption during fasting times. For example, during weekly phone calls, some participants stated they ate snacks in the evening before bed but did not record this in their eating window. Additionally, low-calorie foods were often confused with no-calorie foods, and not recorded even after the interventionist reiterated that any food item with calories must be documented. One participant misunderstood the regimen and only consumed one meal per day throughout the study, which was not revealed until the exit interview. Additionally, five participants thought that TRF was being examined solely for weight loss despite the interventionist explaining that other health outcomes were being evaluated. Due to this misunderstanding, two participants focused on consuming small or low-calorie foods during the eating window rather than eating as normal.

Six of nine participants agreed that they could adhere to the TRF eating pattern for six months, however, this number gradually decreased when asked about the feasibility of maintaining a TRF eating pattern for 12 or 24 months. Six of the participants agreed that fasting became easier over the study period, while two disagreed and one neither agreed nor disagreed. Five of the nine participants stated that they would continue fasting after concluding the study, while two had no preference and two indicated they would not continue. Participant responses to the ten questions within the psychological domain of the Diet Satisfaction Survey are displayed in Figure S4.

#### *3.3. Socio-Environmental Factors*

Family support was central to participant adherence. Although not formally enrolled in the trial, the spouses of participants often altered their eating patterns to accompany their partner. Seven participants were able to space out their meals during their chosen eight-hour eating window rather than consuming all daily calories in one sitting, with four participants reporting inconvenience in eating all of their meals during the eating window. Environmental challenges to TRF adherence included social events during which food was served outside the participant's eating window, as well as changes in work schedules that interfered with the timing of meals. Regular doctor appointments for the participant or their spouse requiring long commutes also extended fasting times beyond the 14-18 hour goal for a few individuals. Additionally, several participants reported that they would not want to follow this eating pattern during holidays or vacations. Notwithstanding these challenges, only one participant stated they would not recommend TRF to a friend, and five believed people would follow this eating pattern if recommended by their healthcare provider. Participant responses to the seven questions within the socio-environmental domain of the Diet Satisfaction Survey are displayed in Figure S5.


analyzed (Figure 5). Plants in LD13 + 4B had the greatest expression levels of *PHYA* and *PHYB*, *3.2. Flowering and Gene Expression*

The flowering percentage of plants was 100% in all treatments (Table 2 and Figure 4). The fastest flowering induction was observed for plants in the control (SD10). It is noteworthy that plants in LD13 + 4B and LD13 + NI − 4B flowered, despite the fact that chrysanthemum is a qualitative SD plant (Table 2 and Figure 4).

**Table 2.** The effects of the supplementary and night-interrupting 10 µmol·m−2 ·s −1 PPFD B light on the flowering characteristics of chrysanthemum (*D. grandiflorum* 'Gaya Yellow'), after 41 days of exposure to the photoperiodic light treatments. flowering characteristics of chrysanthemum (*D. grandiflorum* 'Gaya Yellow'), after 41 days of exposure to the photoperiodic light treatments.

**Table 2.** The effects of the supplementary and night-interrupting 10 μmol·m−2·s−1 PPFD B light on the

*Plants* **2020**, *9*, x FOR PEER REVIEW 6 of 11

followed by plants in SD10 + NI-4B (Figure 5). *PHYA* had the lowest expression levels in plants in LD13 + NI-4B (Figure 5). *PHYB* was the least expressed in plants in LD13 + NI-4B (Figure 5). Plants in LD13 + NI-4B had significantly higher *CRY1* expression levels compared to plants in the other

z See Figure 2 for details on the photoperiodic treatments with B light. y Days of treatment to visible flower bud or days to visible buds. x Mean separation within columns by Duncan's multiple range test at a 5% level. \*\*\*: Significant at *p* ≤ 0.001. z See Figure 2 for details on the photoperiodic treatments with B light. y Days of treatment to visible flower bud or days to visible buds. x Mean separation within columns by Duncan's multiple range test at a 5% level. \*\*\*: Significant at *p* ≤ 0.001.

**Figure 4.** The effects of the supplementary and night-interrupting 10 μmol·m−2·s−1 PPFD B light on the flowering of chrysanthemum (*D. grandiflorum* 'Gaya Yellow'), after 41 days of exposure to the photoperiodic light treatments: side view (**A**) and top view (**B**) (see Figure 2 for details on the photoperiodic treatments with B light). **Figure 4.** The effects of the supplementary and night-interrupting 10 µmol·m−2 ·s −1 PPFD B light on the flowering of chrysanthemum (*D. grandiflorum* 'Gaya Yellow'), after 41 days of exposure to the photoperiodic light treatments: side view (**A**) and top view (**B**) (see Figure 2 for details on the photoperiodic treatments with B light).

Plants in SD10 had the smallest DVB whereas plants in LD13 + NI − 4B had the greatest DVB (Table 2). The DVB was observed to increase as the photoperiod increased (Table 2). The DVB of plants in LD13 + 4B was smaller than those of plants in LD13 + NI − 4B. Interestingly, plants in LD13 + 4B had 93% more flowers per plant compared to plants in the SD10 control. Plants in the SD10 control and SD9 + 4B had the lowest number of flowers (Table 2). Plants in the SD10 control had the greatest flower width (Table 2).

**Figure 5.** The effects of the supplementary and night-interrupting 10 μmol·m−2·s−1 PPFD B light on the relative gene expression levels of) *D. grandiflorum* 'Gaya Yellow' determined by real-time PCR of The photoreceptor gene expression (*PHYA*, *PHYB*, and *CRY1*) in response to the B light was also analyzed (Figure 5). Plants in LD13 + 4B had the greatest expression levels of *PHYA* and *PHYB*, followed by plants in SD10 + NI − 4B (Figure 5). *PHYA* had the lowest expression levels in plants in LD13 + NI − 4B (Figure 5). *PHYB* was the least expressed in plants in LD13 + NI − 4B (Figure 5). Plants in LD13 + NI − 4B had significantly higher *CRY1* expression levels compared to plants in the other treatments (Figure 5). *CRY1* was the least expressed in plants in LD13 + 4B (Figure 5).

*PHYA* (**A**), *PHYB* (**B**), and *CRY1* (**C**). (Details of the NI light qualities are presented in Figure 2). At each sampling date, the individual gene expression levels for the plants in the photoperiodic light treatments were divided by the mean gene expression level for plants in the SD10 control. The data

**Figure 4.** The effects of the supplementary and night-interrupting 10 μmol·m−2·s−1 PPFD B light on the flowering of chrysanthemum (*D. grandiflorum* 'Gaya Yellow'), after 41 days of exposure to the

followed by plants in SD10 + NI-4B (Figure 5). *PHYA* had the lowest expression levels in plants in LD13 + NI-4B (Figure 5). *PHYB* was the least expressed in plants in LD13 + NI-4B (Figure 5). Plants in LD13 + NI-4B had significantly higher *CRY1* expression levels compared to plants in the other

**Table 2.** The effects of the supplementary and night-interrupting 10 μmol·m−2·s−1 PPFD B light on the flowering characteristics of chrysanthemum (*D. grandiflorum* 'Gaya Yellow'), after 41 days of exposure

SD9 + 4B 100 17.7 c x 11.0 c 2.6 b LD13 + 4B 100 22.5 b 21.3 a 0.7 c LD13 + NI-4B 100 28.7 a 15.3 b 0.5 d SD10 + NI-4B 100 18.0 c 15.0 b 2.8 a SD10 100 17.5 c 11.0 c 2.9 a *F*-test \*\*\* \*\*\* \*\*\* z See Figure 2 for details on the photoperiodic treatments with B light. y Days of treatment to visible flower bud or days to visible buds. x Mean separation within columns by Duncan's multiple range

**(%) DVB y (Day) No. of Flowers/Plant Flower width (cm)**

treatments (Figure 5). *CRY1* was the least expressed in plants in LD13 + 4B (Figure 5).

to the photoperiodic light treatments.

**Treatment z Flowering**

test at a 5% level. \*\*\*: Significant at *p* ≤ 0.001.

**Figure 5.** The effects of the supplementary and night-interrupting 10 μmol·m−2·s−1 PPFD B light on the relative gene expression levels of) *D. grandiflorum* 'Gaya Yellow' determined by real-time PCR of *PHYA* (**A**), *PHYB* (**B**), and *CRY1* (**C**). (Details of the NI light qualities are presented in Figure 2). At each sampling date, the individual gene expression levels for the plants in the photoperiodic light treatments were divided by the mean gene expression level for plants in the SD10 control. The data **Figure 5.** The effects of the supplementary and night-interrupting 10 µmol·m−2 ·s −1 PPFD B light on the relative gene expression levels of) *D. grandiflorum* 'Gaya Yellow' determined by real-time PCR of *PHYA* (**A**), *PHYB* (**B**), and *CRY1* (**C**). (Details of the NI light qualities are presented in Figure 2). At each sampling date, the individual gene expression levels for the plants in the photoperiodic light treatments were divided by the mean gene expression level for plants in the SD10 control. The data are presented as the mean ± S.E of the 3 biological replicates. Means accompanied by different letters indicate significant differences (*p* < 0.05), according to Tukey's studentized range test at a 5% significance level.


**6. Age-related changes in intestine intraepitherial lymphocyte subsets and their functional preservation by** *Spirulina* **in mice**

Age-related immune dysfunction has been reviewed by many researchers (Solana et al., 2006). The complex age-related changes in the immune system, collectively termed "immunosenescence," have been demonstrated in diverse species, including humans, and have been recognized as contributing to morbidity and mortality due to greater incidence of infectious diseases, autoimmune diseases, and cancer. The concept of age-related immunosenescence is in agreement with numerous data such as the change of cytokine balances, the decrease of interleukin (IL)-2 contrary to the increase of IL-6, and nutritional imbalance or malnutrition (Miquel, 2001; De la Fuente, 2002). It was reported that antigenspecific secretory immunoglobulin A titer in the intestinal lumen declined in senescent animals (Koga et al., 2000). Some studies have also reported that reduced bioavailability of key conditionally essential nutrients might limit immune response in aging (Cunningham-Rundles, 2004) and that well-nourished elderly people appear to have less significant or minimal changes in immune response (Krause et al., 1999).

It is generally accepted that the development of age-associated alterations occurs earlier in the mucosal immune system than in the systemic immune compartment (Schmucker et al., 2003). The mucosal immune system of the intestinal epithelia contains a functionally

specialized T-cell population known as intraepithelial lymphocytes (IELs). Because of their unique location in the mucosal epithelium, IELs are recognized as a first-line mucosal barrier against infectious diseases and food-borne allergens (Hayday et al., 2001).

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 139

CD45+TCRαβ+ cells were observed in the aged mice, whereas neither an increase nor a decrease was observed in the aged-SP group fed with SpHW—that is, the levels were similar to those in adult mice. In particular, the proportions of CD45+CD8+ cells and CD45+TCRγδ+ cells in the aged-SP group significantly increased in comparison to the aged group. CD8+ T cells expressing αβTCR (αβT cells) are engaged in antigen-specific cell cytotoxicity mediated by major histocompatibility complex (MHC) molecules, whereas T cells expressing γδTCR (γδT cells) often manifest preliminary target cell killing without MHC restriction (Cruse and Lewis, 1995). γδT cells have also been shown to be associated with regulation of the generation and differentiation of IELs (Komano et al., 1995). These results suggest that ingestion of SpHW in the aged-SP group may contribute to the functional preservation of the intestinal epithelium as a first line of mucosal barrier against

Decreased levels of RBCs, especially the level of hematocrit, Ht, in the aged group, were also restored after ingestion of SpHW in the aged-SP group. Significant decreases in WBCs in the aged-SP group, in contrast to the increase in the aged group, may be ascribed to the antiinflammatory activity of *Spirulina* (Vila et al., 2008) and/or to the restoration of immunological function by ingesting *Spirulina*. Some reports indicated that phycocyanin and the polysaccharide isolated from *Spirulina* increased bone marrow nucleated cell and erythrocyte counts in the gamma-ray irradiated mice or dog (Zhang, 1994; Zhang et al., 2001; Verma et al., 2006). Many studies have demonstrated that *Spirulina* including phycocyanin possesses antioxidant activity, as well as an anti-inflammatory activity (Romay et al., 1998; Remirez et al., 2002), which scavenges peroxyl radicals, and also acts as an inhibitor of cyclooxygenase, like nonsteroidal anti-inflammatory drugs. In addition, a downregulation of pro-inflammatory cytokines, such as TNF-α and -γ, was observed in the aged animals on the *Spirulina*-enriched diet (Vila et al., 2008). Overexpression of MHC class Irelated chain A in the intestine of experimental transgenic mice resulted in a clonal expansion of CD4+CD8αα+ IELs and attenuated acute colitis in an experimental model of inflammatory bowel disease induced by dextran sodium sulfate administration (Park et al., 2003). CD8αα+ IELs developed along an extrathymic pathway may work as antiinflammatory regulator T cells to sustain the mucosal intranet formed by intestinal epithelial cells and IELs and to diminish the expansion of enterotoxigenic *Escherichia coli* (Kim et al., 2001). Although ingesting SpHW did not significantly increase the level of CD4+CD8+ IELs in the present study, these facts, in addition to our present results, suggest that ingestion of *Spirulina* appears to be effective for protecting immune functions or improving immune systems vulnerable to age, thereby reducing the risk of infectious and autoimmune diseases.

*Spirulina* and its extracts enhanced proliferation of hematopoietic cells and colony formation of bone marrow cell, as a marker for cell differentiation activity, in *in-vitro* and *in-vivo* study using mice. Phycocyanin, a light-harvesting pigment of *Spirulina*, also induced cell differentiation of human leukemia cell lines, U937 and HL-60 cells, into monocyte/macrophage

infectious agents through retaining the numbers of certain IELs.

However, additional detailed study is needed.

**7. Conclusions**

We have reported that ingestion of phycocyanin enhanced the antigen-specific immunoglobulin A response in the intestinal mucosa of mice (Nemoto-Kawamura et al., 2004). In this section, we investigated age-related changes in intestine IEL subsets in mice by flow cytometric (FCM) analysis and their functional preservation after the animals were fed *Spirulina*.

#### **Characterization of IELs of adult and aged mice**

IELs possess phenotypic features distinct from those of lamina propria lymphocytes in intestine. Lamina propria lymphocytes consist of predominantly activated T cells and are mainly CD4+ and CD8+ single-positive T cells in proportions of about 70% and 30%, respectively. The phenotype of lamina propria lymphocytes, in general, is similar to that of the cells in the peripheral lymphoid tissues and in the circulating blood, that is, over 95% of the cells possess a surface phenotype of T-cell receptor αβ+ (TCRαβ+), whereas less than 5% possess TCRγδ+. These cells are known to be matured in the thymus (Lydyard and Grossi, 1998). IELs, on the other hand, possess TCRγδ+ in a greater percentage (30–60%) and TCRαβ+ in a percentage of 40–70%, which might be related to their state of activation (Ewijk et al., 1999). In adult mice bred in a conventional environment, about half of the IELs have a phenotype of surface CD antigen similar to that of most peripheral T lymphocytes, that is, Thy-1+, TCRαβ+, and either CD4+ or CD8+, which are made up of heterodimers of CD8α and β chains (CD8αβ+). These cells were matured in a thymus-dependent manner (Kaminogawa and Nanno, 2004). Another major IEL population possesses the surface phenotype TCRαβ+ or TCRγδ+, which expresses CD8 homodimeric α chains (CD8αα+) but does not express CD4 or CD8 heterodimeric molecules (Rocha et al., 1994). These cells are known to be of extrathymic origin. Small percentages of the TCRαβ+ and TCRγδ+ but no TCR cells are CD8– CD4–. TCRαβ+ IELs co-expressing both CD4 and CD8 molecules are rare but bear high levels of TCRαβ and CD8αα (Lefrancois, 1991). Our preliminary experiment showed that the number of CD45+ (leukocyte-common antigen-positive) cells as IELs was significantly lower in aged mice than in adult mice. Either the proportion or the number of CD8+ cells in addition to CD45+ cells of aged mice was significantly lower than that of adult mice, corresponding to the previous article by Komuro et al (1990). The proportion and number of CD4+CD8+ double-positive cells in the aged mice, on the other hand, were higher than those in adult mice. It has been reported that CD4+CD8+ T cells bearing TCRαβ in the epithelium, which were derived from thymus-dependent populations, expanded with aging at a local site of the intestine under the influence of intestinal microflora, contributing to the first line of defensive barrier in the epithelium (Takimoto et al., 1992).

Overall, increased or decreased levels of these surface antigen-positive cells observed in the aged mice tended to be restored by the ingestion of SpHW for 5 weeks in the aged-SP group. In fact, significant decreases of CD45+CD8+ cells and increases of CD8–CD4– and CD45+TCRαβ+ cells were observed in the aged mice, whereas neither an increase nor a decrease was observed in the aged-SP group fed with SpHW—that is, the levels were similar to those in adult mice. In particular, the proportions of CD45+CD8+ cells and CD45+TCRγδ+ cells in the aged-SP group significantly increased in comparison to the aged group. CD8+ T cells expressing αβTCR (αβT cells) are engaged in antigen-specific cell cytotoxicity mediated by major histocompatibility complex (MHC) molecules, whereas T cells expressing γδTCR (γδT cells) often manifest preliminary target cell killing without MHC restriction (Cruse and Lewis, 1995). γδT cells have also been shown to be associated with regulation of the generation and differentiation of IELs (Komano et al., 1995). These results suggest that ingestion of SpHW in the aged-SP group may contribute to the functional preservation of the intestinal epithelium as a first line of mucosal barrier against infectious agents through retaining the numbers of certain IELs.

Decreased levels of RBCs, especially the level of hematocrit, Ht, in the aged group, were also restored after ingestion of SpHW in the aged-SP group. Significant decreases in WBCs in the aged-SP group, in contrast to the increase in the aged group, may be ascribed to the antiinflammatory activity of *Spirulina* (Vila et al., 2008) and/or to the restoration of immunological function by ingesting *Spirulina*. Some reports indicated that phycocyanin and the polysaccharide isolated from *Spirulina* increased bone marrow nucleated cell and erythrocyte counts in the gamma-ray irradiated mice or dog (Zhang, 1994; Zhang et al., 2001; Verma et al., 2006). Many studies have demonstrated that *Spirulina* including phycocyanin possesses antioxidant activity, as well as an anti-inflammatory activity (Romay et al., 1998; Remirez et al., 2002), which scavenges peroxyl radicals, and also acts as an inhibitor of cyclooxygenase, like nonsteroidal anti-inflammatory drugs. In addition, a downregulation of pro-inflammatory cytokines, such as TNF-α and -γ, was observed in the aged animals on the *Spirulina*-enriched diet (Vila et al., 2008). Overexpression of MHC class Irelated chain A in the intestine of experimental transgenic mice resulted in a clonal expansion of CD4+CD8αα+ IELs and attenuated acute colitis in an experimental model of inflammatory bowel disease induced by dextran sodium sulfate administration (Park et al., 2003). CD8αα+ IELs developed along an extrathymic pathway may work as antiinflammatory regulator T cells to sustain the mucosal intranet formed by intestinal epithelial cells and IELs and to diminish the expansion of enterotoxigenic *Escherichia coli* (Kim et al., 2001). Although ingesting SpHW did not significantly increase the level of CD4+CD8+ IELs in the present study, these facts, in addition to our present results, suggest that ingestion of *Spirulina* appears to be effective for protecting immune functions or improving immune systems vulnerable to age, thereby reducing the risk of infectious and autoimmune diseases. However, additional detailed study is needed.

#### **7. Conclusions**

138 Blood Cell – An Overview of Studies in Hematology

**Characterization of IELs of adult and aged mice**

of defensive barrier in the epithelium (Takimoto et al., 1992).

Overall, increased or decreased levels of these surface antigen-positive cells observed in the aged mice tended to be restored by the ingestion of SpHW for 5 weeks in the aged-SP group. In fact, significant decreases of CD45+CD8+ cells and increases of CD8–CD4– and

*Spirulina*.

specialized T-cell population known as intraepithelial lymphocytes (IELs). Because of their unique location in the mucosal epithelium, IELs are recognized as a first-line mucosal

We have reported that ingestion of phycocyanin enhanced the antigen-specific immunoglobulin A response in the intestinal mucosa of mice (Nemoto-Kawamura et al., 2004). In this section, we investigated age-related changes in intestine IEL subsets in mice by flow cytometric (FCM) analysis and their functional preservation after the animals were fed

IELs possess phenotypic features distinct from those of lamina propria lymphocytes in intestine. Lamina propria lymphocytes consist of predominantly activated T cells and are mainly CD4+ and CD8+ single-positive T cells in proportions of about 70% and 30%, respectively. The phenotype of lamina propria lymphocytes, in general, is similar to that of the cells in the peripheral lymphoid tissues and in the circulating blood, that is, over 95% of the cells possess a surface phenotype of T-cell receptor αβ+ (TCRαβ+), whereas less than 5% possess TCRγδ+. These cells are known to be matured in the thymus (Lydyard and Grossi, 1998). IELs, on the other hand, possess TCRγδ+ in a greater percentage (30–60%) and TCRαβ+ in a percentage of 40–70%, which might be related to their state of activation (Ewijk et al., 1999). In adult mice bred in a conventional environment, about half of the IELs have a phenotype of surface CD antigen similar to that of most peripheral T lymphocytes, that is, Thy-1+, TCRαβ+, and either CD4+ or CD8+, which are made up of heterodimers of CD8α and β chains (CD8αβ+). These cells were matured in a thymus-dependent manner (Kaminogawa and Nanno, 2004). Another major IEL population possesses the surface phenotype TCRαβ+ or TCRγδ+, which expresses CD8 homodimeric α chains (CD8αα+) but does not express CD4 or CD8 heterodimeric molecules (Rocha et al., 1994). These cells are known to be of extrathymic origin. Small percentages of the TCRαβ+ and TCRγδ+ but no TCR cells are CD8– CD4–. TCRαβ+ IELs co-expressing both CD4 and CD8 molecules are rare but bear high levels of TCRαβ and CD8αα (Lefrancois, 1991). Our preliminary experiment showed that the number of CD45+ (leukocyte-common antigen-positive) cells as IELs was significantly lower in aged mice than in adult mice. Either the proportion or the number of CD8+ cells in addition to CD45+ cells of aged mice was significantly lower than that of adult mice, corresponding to the previous article by Komuro et al (1990). The proportion and number of CD4+CD8+ double-positive cells in the aged mice, on the other hand, were higher than those in adult mice. It has been reported that CD4+CD8+ T cells bearing TCRαβ in the epithelium, which were derived from thymus-dependent populations, expanded with aging at a local site of the intestine under the influence of intestinal microflora, contributing to the first line

barrier against infectious diseases and food-borne allergens (Hayday et al., 2001).

*Spirulina* and its extracts enhanced proliferation of hematopoietic cells and colony formation of bone marrow cell, as a marker for cell differentiation activity, in *in-vitro* and *in-vivo* study using mice. Phycocyanin, a light-harvesting pigment of *Spirulina*, also induced cell differentiation of human leukemia cell lines, U937 and HL-60 cells, into monocyte/macrophage

and granulocyte, respectively, to some extent directly and indirectly through enhancing cytokine production in human peripheral blood lymphocytes stimulated with phycocyanin. These distinguished activities of *Spirulina* as well as other certain functional foods can be preferably emphasized to be used, especially for elderly people. Recent intervention study showed that 6- and 12-week supplementation of *Spirulina* increased mean hemoglobin level and indoleamine 2,3-dioxygenase activity, as a sign of immune function, in the elderly subjects, suggesting that *Spirulina* may ameliorate anemia and immunosenescence in elderly people (Selmi et al., 2011). Pentón-Rol et al. (2011) demonstrated that phycocyanin triggered preventing or downgrading experimental autoimmune encephalitis (EAE) expression in rats, and that ingestion of phycocyanin induced a regulatory T cell (Treg) response in peripheral blood mononuclear cells from the patients with multiple sclerosis (MS). The authors suggested that phycocyanin may act as a neuroprotector and thereby may restore the functional damage in neurodegenerative disorders of the central nervous system (CNS). Another animal model in rats showed that *Spirulina* promoted stem cell genesis and protected against LPS-induced declines in neural stem cell proliferation, and that cytokines did appear capable of regulating several phases of the neurogenesis process, supporting their hypothesis that a diet enriched with *Spirulina* may help protect the stem/progenitor cells from insults (Bachstetter et al., 2010). These studies including reports summarized in this chapter show that *Spirulina* is useful in providing complementary nutrients for modulating or maintaining the immune system and that is also may have potential therapeutic benefits for improvement of immune dysfunctions caused by, for example, radiation, chemotherapy using anti-cancer and anti-infectious drugs, and certain microorganisms such as human immunodeficiency virus (HIV) itself, other than ageing. Further research along these lines is needed to validate these evidences.

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 141

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#### **Author details**

Osamu Hayashi *Kagawa Nutrition University, Japan*

#### **8. References**

Balachandran, P, Pugh, ND, Ma, G, and Pasco, DS (2006). "Toll-like receptor 2-dependent activation of monocytes by Spirulina polysaccharide and its immune enhancing action in mice", *Int Immunopharmacol*, 6, 1808-1814.

140 Blood Cell – An Overview of Studies in Hematology

**Author details**

*Kagawa Nutrition University, Japan*

Osamu Hayashi

**8. References**

and granulocyte, respectively, to some extent directly and indirectly through enhancing cytokine production in human peripheral blood lymphocytes stimulated with phycocyanin. These distinguished activities of *Spirulina* as well as other certain functional foods can be preferably emphasized to be used, especially for elderly people. Recent intervention study showed that 6- and 12-week supplementation of *Spirulina* increased mean hemoglobin level and indoleamine 2,3-dioxygenase activity, as a sign of immune function, in the elderly subjects, suggesting that *Spirulina* may ameliorate anemia and immunosenescence in elderly people (Selmi et al., 2011). Pentón-Rol et al. (2011) demonstrated that phycocyanin triggered preventing or downgrading experimental autoimmune encephalitis (EAE) expression in rats, and that ingestion of phycocyanin induced a regulatory T cell (Treg) response in peripheral blood mononuclear cells from the patients with multiple sclerosis (MS). The authors suggested that phycocyanin may act as a neuroprotector and thereby may restore the functional damage in neurodegenerative disorders of the central nervous system (CNS). Another animal model in rats showed that *Spirulina* promoted stem cell genesis and protected against LPS-induced declines in neural stem cell proliferation, and that cytokines did appear capable of regulating several phases of the neurogenesis process, supporting their hypothesis that a diet enriched with *Spirulina* may help protect the stem/progenitor cells from insults (Bachstetter et al., 2010). These studies including reports summarized in this chapter show that *Spirulina* is useful in providing complementary nutrients for modulating or maintaining the immune system and that is also may have potential therapeutic benefits for improvement of immune dysfunctions caused by, for example, radiation, chemotherapy using anti-cancer and anti-infectious drugs, and certain microorganisms such as human immunodeficiency virus (HIV) itself, other than

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**Chapter 8**

© 2012 Selz, licensee InTech. This is an open access chapter 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.

The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2012 Selz, licensee InTech. This is a paper 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.

**Spontaneous Alternation Behavior**

**SAB:** Spontaneous alternation behavior (SAB) generally refers to the tendency of animals, even single-celled organisms, to alternate their non-reinforced (Dember & Richman, 1989) choices of T- or Y-maze arms on subsequent trials, following an initial trial or turn. First described over 80 years ago (Tolman, 1925), the phenomenon has been ascribed to the operation of a variety of mechanisms including Hullian reactive inhibition (Solomon, 1948), stimulus satiation (Glanzer, 1953), action decrement (Walker, 1958), curiosity (Dember and Earl, 1957), habituation to novelty (Carlton, 1969), foraging strategies (Estes and Schoeffler, 1955) and spatial working memory (Sarter, et al., 1988). Studies have suggested that the primary cue for alternation among invertebrates to be is the body turn. Vertebrates rely primarily on directional and odor cues. The fitness benefits associated with stimulus seeking and behavioral exploration, foraging, remain the most compelling explanation of why SAB is found ubiquitously and reliably (Richman, et al.1986). Although the underlying mechanism of SAB is open to study, there is general agreement that the ability to alternate

choices requires that the organism remember its previous choice (Hughes, 2004).

Because SAB implicates future behavior which is statistically dependent on prior behavior and accompanied by memorially-dependant loss of degrees of freedom, SAB has been used to suggest the presence of a functional short-term memory. A left or right turn in a T- or Ymaze is a statistical function of the presence and direction of the previous turn, when such a

While theories of memory are many and not the focus of this chapter, SAB is generally suggested to be recent memory dependent in animals complex enough to possess the structures postulated to underlie recent memory, and to be dependent on a more basic sensory/membrane/receptor depletion time-limited memorial mechanism in simple

and reproduction in any medium, provided the original work is properly cited.

**in Human Neutrophils**

Additional information is available at the end of the chapter

Karen A. Selz

http://dx.doi.org/10.5772/47851

**1. Introduction**

prior turn exists.


2. TDS basis

The pioneer publication on the TDS technique, [5], explains in detail the derivation of the equations. The Laplace space solution of the arithmetic pressure derivative for a homogeneous and infinite reservoir with skin and wellbore storage is also presented in [5] and given by

$$P\_D ' = \frac{4}{\pi^2} \int\_0^\infty \left( \frac{e^{-u^2 t\_D}}{u \left\{ \left[ u C\_D I\_0(u) - (1 - C\_D u u^2) I\_1(u) \right]^2 + \left[ u C\_D Y\_0(u) - (1 - C\_D u u^2) Y\_1(u) \right]^2 \right\}} \right) du \,\tag{1}$$

However, we know that the pressure derivative is a horizontal line during radial flow regime. The dimensionless pressure derivative during radial line is easier represented by

$$t\_D \, ^\*P\_D '= \mathbf{0}.\mathbf{5}.\tag{2}$$

Then, to obtain practical equations, dimensionless parameters must be used. The dimensionless time, based upon half-fracture length and reservoir drainage area, is given below:

$$t\_{D\text{xf}} = \frac{0.000263kt}{\phi \mu c\_t \varkappa\_f^2} \tag{3}$$

and

$$t\_{DA} = \frac{0.000263kt}{\phi \mu c\_t A}.\tag{4}$$

The dimensionless pressure and pressure derivative parameters for oil reservoirs are given by

$$P\_D = \frac{kh\Delta P}{141.2q\mu B} \tag{5}$$

and

$$t\_D \, ^\ast P\_D \, ^\prime = \frac{kh \, (t \, ^\ast \Delta P \, ^\prime)}{141.2 q \mu B} \, . \tag{6}$$

Finally, the dimensionless fracture conductivity introduced in [3] is defined as

$$\mathbf{C}\_{f\mathbf{D}} = \frac{k\_f w\_f}{k\_\cdot \mathbf{x}\_f}.\tag{7}$$

It is observed from Eq. (5) that the two key parameters of a hydraulic fracture are the half-fracture length, xf, and the fracture conductivity, kf wf. The total length of the fracture is given by 2 xf.

The easiest application of TDS technique is given by replacing the dimensionless pressure derivative defined by Eqs. (6) and (2), to provide an expression to readily determine formation permeability:

$$k = \frac{70.6q\mu B}{h(t\*\Delta P')\_R},\tag{8}$$

where (t\*ΔP0 )R is the pressure derivative value during radial flow regime. The equations for the TDS technique are derived in the same manner Eq. (8) was obtained.


**6. Impacts of climate change on crop production**

Due to climatic changes overall crop production system has affected, ultimately causing a challenge to global food security. But the more severe impact of these changes has been observed in underdeveloped countries. Over the next decade it is predicted that billions of people, particularly from underdeveloped countries may encountered with water as well as food scarcity, accompanied with a high risk to the life and health due to climate changes. Developing countries are more prone to the changing climatic conditions as these countries lack in social, financial as well as technological resources, which required facing the climate change [17].

Environmental conditions always cast an impact on either the succession or failure of crops, while the management of stresses caused due to these changes has been part of multidisciplinary studies. Global crop production system has shown continuous susceptibility to the risks of changing climatic conditions. Now farmers have been facing severe challenges than the normally experienced, due to changing climatic conditions. Global climatic conditions became extreme like, warmer temperatures, increased coastal waters, heavy precipitation, and geographical shifts in drought as well as storm patterns [18].

It is estimated that climatic changes may cause a considerable decrease in maize production in southern Africa. It may also cause up to 10% decrease in staple crops of south Asia, including rice while more than 10% decrease in millet and maize production [19]. With a slight increase of 1-3°C in the mean local temperature of some moderate- to high latitude areas, productivity may also be increased, depending on

#### **Figure 3.**

*Food chain from pre-production to consumption.*

#### **Table 1.**

*Impacts of climate change on crop production [22].*

the crop. In contrasting, in areas of lower latitudes productivity of crop decreased with the even slightest change in relative temperature range [20].

Unpredictable seasonal as well annual, fluctuations have been observed in crop production system due to the abrupt outbreaks of disease and pest and other extreme events. This require an efficient adaptable management response towards these changing scenarios [21].

Agriculture crop production is facing a number of impacts due to climate change in the components of weather/climate such as temperature, precipitation, cyclones, sea level etc. (**Table 1**).

#### **7. Modern tools of climate smart crop production**

Climate change, which includes high temperatures and drought, is projected to have a detrimental effect on plant agronomic conditions as well as soil nutrients, diseases, and pests. As a result, climate-resilient varieties with broad spectrum and long-term tolerance to both biotic and abiotic stresses are required. The new genetic engineering method for crop enhancement is precise genome editing [23]. Climate change has put a pressure on researcher, farmers and scientists working in the field of agriculture to adopt new technologies to cope with the prevailing issues (**Figure 4**). For targeted genome editing in plants, several techniques have been developed, including zinc finger nucleases (ZFNs), TAL effector proteins (TALENs), RNA directed nucleases (RGENs), and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9. Both of these approaches depend on the creation of double stranded breaks at particular loci and the activation of the DNA repair system [24].

#### **7.1 Genome editing**

Crops with higher yields and greater resistance to abiotic stress are needed to meet the demands of a growing global population and the effect of climate change on agriculture. Traditional crop improvement through genetic recombination or random mutagenesis, on the other hand, is a time-consuming process that cannot keep up with rising crop demand. Genome editing techniques including clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) allow for selective alteration of almost any crop genome

*Climate Smart Crops for Food Security DOI: http://dx.doi.org/10.5772/intechopen.99164*

**Figure 4.** *Impact of climate change on agriculture.*

sequence to generate novel variation and speed up breeding efforts. We anticipate a gradual transition away from conventional breeding and toward selective genome editing cycles in crop improvement. Crop enhancement by genome editing is not limited by existing variation or the need to pick alleles through several breeding generations. However, the lack of full reference genomes, a lack of awareness of possible modification goals, and the legal status of edited crops restrict current crop genome editing applications. We believe that overcoming the technological and social barriers to genome editing's implementation will allow this technology to produce a new generation of high-yielding, climate-ready crops [25]. At our lab, we are using different online platforms such as CHOPCHOP, CRISPR-P, MultiTargetor etc, and reagents provided by Addgene, Vectorbuilder, GeneCopoeia, Nootropics Frontline etc. for genome editing in crops.


**4. Conclusions**

In summary, the relatively conserved elements of the viral life cycle offer many opportunities to reexamine compounds developed to address previous outbreaks for efficacy against novel outbreaks. Clear examples are shown in the results against non-structural proteins above, which often maintain significantly higher sequence homology across species due to a conserved mechanism than structural proteins. However, while this sequence conservation indeed leads to a degree of "cross-talk" between nsp inhibitors, the required exquisite and intricate nature of the interaction between a successful drug and its target will almost inevitably reduce the efficacy of a monotherapy. As a result, it is likely that while repurposing can identify promising candidates, care must be taken not to hope for a single effective solution in existing drugs (e.g. Ivermectin, hydroxychloroquinine, etc.). Rather, functional (clinical) solutions are much more likely be found in careful clinical trials of combination therapies of drugs identified through repurposing screens.

The current combination of virtual, *in vitro* and *in vivo* screening is well positioned to perform rapid repurposing experiments on the relatively small number of clinically approved candidate molecules. However, significant research effort should be expended globally to continue to identify potential viral inhibitors and further populate the potential repurposing list.

Response speed is a key factor facing outbreaks. Drug repurposing is a practical solution that provides multiple benefits beyond classical drug discovery. Perhaps the greatest advancement in this area has been the improvements in computational techniques, that has developed in parallel with advances in structural biology—both of which continue to improve. These structural views of the proteins driving disease expand the number of experiments that can be performed *in silico*, providing both an increase in speed of hypothesis generation, as well as an important pre-filter stage to select out candidate molecules for screening. In our opinion a key aspect that should not be overlooked is the *in vitro* validation of a molecular effect on a proposed target. Certain recent experiences have shown that attempts to bypass this stage and short-cut the process by directly jumping into clinical trials can produce conflicting results, leading to confusion and loss of confidence of the public. However, despite successful application of drug repurposing, no single golden standard as yet exists to give relatively predictable results.


**Conflict of interest**

The author has no conflict of interest to declare.

*Amino Acids as Neurotransmitters. The Balance between Excitation and Inhibition… DOI: http://dx.doi.org/10.5772/intechopen.103760*


**Table 6.**

*Plant-based vaccines for pandemics and epidemics infectious diseases.*

## *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

cVLP vaccine. CPG adjuvant vaccine was administered in healthy adult subjects in two age groups, Part A (18–49 years) and Part B (50–85 years). The company also produces seasonal influenza vaccines through *Nicotiana benthamiana* plants [86]. BaiyaPharming™ produced a subunit-based plant-derived vaccine against SARS-CoV-2 in *N. benthamiana*. Baiya SARS-CoV-2 Vax 1 was chosen between six candidates which showed better immunogenicity in mice and monkeys. Baiya Phytopharm expects to initiate human trials by September 2021 [87]. Plant-derived vaccines for humans and animals stand out as a viable alternative that can be used to overcome the barriers of conventional vaccines. Within the scope of transgenic plants, it is possible to produce cost-effective, immunogenic and safer vaccines in plants with an enhanced amount, effective isolation and purification methods. Correct use of adjuvants along with the production of recombinant vaccine antigens also seems equally crucial for the future of this technology.

#### **3.2 Replacement human proteins**

Plants are usually referred to as molecular factories to provide humans with many useful molecules for many purposes. In the last decades, it has also become available to produce specific heterologous proteins as a replacement in humans. In this manner, the first plant-derived pharmaceutical was human growth hormone, which was expressed in transgenic tobacco as a fusion with *Agrobacterium* nopaline synthase enzyme in 1986 [88]. Today, recombinant human proteins are a considerable part of FDA-approved biotechnological drugs and recombinant plant-derived proteins are extended in many categories as industrial enzymes, research intended technical proteins, nutritional supplements and polymers as well as antibodies and vaccines which were mentioned in the previous section. Replacement human proteins include products as growth hormone, HSA, α-interferon, erythropoietin (EPO), human secreted alkaline phosphatase, aprotinin, collagen, α1-antitrypsin and more (**Table 7**).

Human growth hormone (hGH) has various biological functions on protein synthesis, cell proliferation and metabolism. After the first successful plantderived production, many different strategies were achieved. In a recent approach, a synthetic hGH gene (shGH) has been synthesized in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter. The plant expression vector was introduced into rice calli (*O. sativa* L.) via the particle bombardment transformation method. The shGH protein expression was verified and quantified as 57 mg/L in the transgenic rice cell suspension medium. Biological activities of the shGH were found similar to the conventional *E. coli*-derived recombinant hGH. Likewise, many different plant tissues and expression systems are suggested as effective hGH production replacements [101].

Human serum albumin (HSA) is the most abundant protein in human blood plasma. HSA is a soluble, globular, unglycosylated, monomeric multidomain protein. The single polypeptide of HSA consists of 585 amino acids with a range of structural configurations that fold into three helical domains [104]. It is also the first full-size native human protein expressed in plants and there is more than 500 tons annual demand. The transient expression level of the HSA gene in different genotypes was achieved in many plants. Recently, the A. tumefaciens strains LB4404 and GV3101 containing pBI121-HSA binary vector were infiltered in *Nicotiana benthamiana* and *N. tabacum* varieties. The bioactive HAS expression in tobacco leaves through the expression of the HSA gene in the plant system is suggested as the first transient expression success in literature [105].

α-interferon is the first human pharmaceutical protein produced in rice. The plasmid pIG3031 containing human α-interferon cDNA and the neomycin

#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

**Table 7.**

*Replacement human proteins produced from transgenic plants.*

*Genetically Modified Plants and Beyond*

**70**

phosphotransferase II coding sequence was introduced to Indica rice protoplasts via lipofection-mediated transformation. Transgenic plants were regenerated from transformed calli. Extracts of transgenic cell cultures and plants presented apparent interferon activity proven by the resistance of human amniotic cell lines to viral infection in the presence of plant extracts. This production encouraged many other strategies as human α-interferon cDNA was correctly expressed in rice cells [106].

Erythropoietin (EPO) is the first human replacement protein produced in tobacco suspension cells. EPO is a cytokine that regulates and maintains the physiological level of circulating erythrocytes. The survival of erythroid precursor cells is also achieved through EPO. It stimulates the proliferation and differentiation of the precursor cells by plasma membrane EPO-receptor interactions. The first transgenic approach introduced human Epo cDNA via *A. tumefaciens*-mediated gene transfer to tobacco BY2 cells (*N. tabacum* L. cv. Bright Yellow 2). EPO is a heavily glycosylated protein, therefore glycosylation of tobacco-derived EPO by smaller oligosaccharides led the molecule to remain attached to the cell wall. However, it induced the differentiation and proliferation of erythroid cells in in vitro biological activity trials [107]. Recently, mammalian cell-derived recombinant human erythropoietin (rhuEPOM) is a multimodal neuroprotectant in experimental stroke models. However, the rhuEPOM clinical trials were terminated due to the increased risk of thrombosis, largely ascribed to its erythropoietic function. A rhuEPO derivative without sialic acid residues was produced in a plant-based expression system which is lacking sialylation capacity to produce asialo-rhuEPOP . Repeated intravenous injection (44 μg/kg bw) in mice presented no increase in hemoglobin levels and red blood cells. Hence, Asialo-rhuEPOP that lacks erythropoietic activity and immunogenicity suggested as a great multimodal neuroprotectant for stroke treatment [108].

Pathogen contamination risk of animal-derived collagen initiated the need for safe recombinant production of this complex molecule. Collagen is the first human structural-protein polymer produced in a plant-derived system. The use of the tobacco plant as a novel expression system for the production of human homotrimeric collagen I was achieved in 2000. cDNA encoding the human proalpha1(I) chain was introduced to tobacco. Expressed recombinant procollagen has been folded to stable homotrimeric triple helix-shaped collagen as in animal cells [109]. *A. tumefaciens*-mediated transient expression of the recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H) improved the quality of collagen by enhancing thermostability to 37°C [110].

Human 1-antitrypsin (AAT) is a 394-amino-acid glycoprotein that inhibits the activity of the serine protease neutrophil elastase. Healthy individuals control elastase activity by producing sufficient quantities of AAT into the bloodstream. When the circulating concentration drops below 15%, various diseases as emphysema, hepatitis and skin disorders occur. Various expression systems, cell types and tissues of plants were used for molecular pharming earlier. However, rice suspension cells were used for the molecular farming of recombinant human 1-antitrypsin (rAAT) in biologically active form for the first time in 1999. Transformation of rice callus tissues with a p3D-AAT expression vector containing the cDNA for mature human AAT protein was achieved. The promoter, signal peptide and terminator of a rice-amylase gene Amy3D, which tightly controls simple sugars such as sucrose, regulated expression and secretion of rAAT. Expression of the rAAT was initiated by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. For that time being, the rice cell culture system clearly contributed to the molecular pharming field.

As a concluding summary, plants have various upsides against traditional microbial and animal cell culture systems in respect to molecular pharming. Some of these include cost efficiency, easier up-scale production, absence of human pathogens and accurate maturation (folding and assembly) of proteins. The potential of molecular pharming in plants attracts more and more entrepreneurs following the numerous successful products and companies. Achieving secretory properties and targeting of plant products also surpass many other production systems. The use of plants as bioreactors is well known, and its applications are increasing for both recombinant protein expression and recombinant pharmaceutical production. This chapter showcases the various plant biotechnology application and strategies as applied in the production of recombinant antibodies, antigens, therapeutic proteins and enzymes, that are used in the treatment of various diseases. Now with the COVID-19 pandemic, more than ever this approach is taking center stage. This is so important as it will reveal the hidden treasure that transgenic plants already offered but mired in the genetically modified organism debate and therefore rejected before these other applications beyond food could be readily realized. Indeed, different systems exist within the system utilizing many different parts and tissues of the plant to produce products. Furthermore, this system is more advantageous in a eukaryotic system that performs post-translational modification as would animal and yeast cells thus yielding the final desired therapeutic product comparable to what is already produced by humans, for example.


**7. Conclusion**

This chapter discusses two different air separation units. Both systems are designed for a small production capacity—260 t/d of gaseous oxygen and 43 t/d of liquid oxygen. The results of the exergetic analysis show an exergetic efficiency of 28.4 and 31.1% for the system with external and internal compression unit, respectively. From the economic point of view, the bare module costs are slightly higher for the system with external compression unit (Case A) in comparison to the system with internal compression unit (Case B). The results for the total revenue requirement show a relatively large contribution of the fuel cost, which leads to a lower *TRR* in Case B in comparison to Case A. In addition, Case B has advantages from the safety point of view due to the use of an oxygen pump instead of an oxygen compressor.


**1. Introduction**

Foods and beverages are essentially complex mixtures of chemicals consumed for either sustenance or pleasure. The diversity of chemicals found in food is vast, as are their varying properties. It has long been known that chemicals with carcinogenic activity in rodent models can be found in many commonly consumed foods [1–5] from a variety of sources including plants, microorganisms, contaminations, additive uses and reactions which occur during storage, processing and cooking [2] (Table 1). In addition, carcinogens can be formed endogenously, from food materials [6–8]. This review focuses mainly on carcinogens, both rodent and human, present in foods and beverages at low concentrations which are imperceptible, and a few components present at levels associated with adverse effects. It does not address drinking water contaminants, such as arsenic, or the contributions of caloric content and macro components such as fat content, or the excess consumption of alcoholic beverages, all of which, nevertheless, have been implicated in increased cancer risks in humans [9–16].

Chemical carcinogens exert their effects through two distinct types of mechanism of action (MoA), which have been characterized as DNA-reactive (genotoxic) and epigenetic (non-genotoxic) [17–20], as discussed below. Chemical structure determines the carcinogenic MoAs; DNA-reactive carcinogens have structures that form reactive electrophiles, either directly or following bioactivation, whereas epigenetic carcinogens lack such properties, but have structures that exert other molecular and cellular effects leading

**Citation:** Kobets, T.; Smith, B.P.C.; Williams, G.M. Food-Borne Chemical Carcinogens and the Evidence for Human Cancer Risk. *Foods* **2022**, *11*, 2828. https://doi.org/10.3390/ foods11182828

Academic Editors: Andreas Eisenreich and Bernd Schaefer

Received: 9 August 2022 Accepted: 8 September 2022 Published: 13 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to cancer [17,18]. These differences in MoA underly the nature of human cancer risks from exposures [21,22].

**Table 1.** Sources of detectable carcinogens in food.

a Many of the agents listed are detectable only at minute levels by highly sensitive analytical techniques.

#### *1.1. Mechanisms of Carcinogenicity of DNA-Reactive Carcinogens*

DNA-reactive carcinogens have structures that permit formation of electrophilic reactants that covalently bind (adduct) to nucleophilic sites in nuclear DNA, as well as in other macromolecules, including RNA and proteins, in the target tissue(s) of carcinogenicity [23–25]. In target tissue(s), a single DNA reactant can form different DNA adducts on various nucleophilic sites either on a single base or on different bases. Each adduct can undergo different rates of repair depending upon its location in the genome. For example, adducts in transcriptionally active regions are repaired by a transcription-coupled repair system whereas adducts in transcriptionally silent regions are repaired by a global repair system [26]. The levels of DNA adducts resulting from exposures are a function of several metrics including dose levels, the frequency of exposure, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it gives rise to mutations, with those at sites of base pairing being more mutagenic.

Pro-mutagenic DNA alterations are converted to mutations during cell replication [27–29]. Mutations in critical growth control genes lead to neoplastic conversion, and subsequent neoplastic development [28,30]. DNA-reactive carcinogens can also exert other cellular effects, such as cytotoxicity, leading to enhanced cell proliferation, which can contribute to their carcinogenic activity [31,32]. DNA-reactive carcinogens can have additive effects with one another in their target organ(s).

Some DNA adducts evidently do not lead to carcinogenicity, since some adducts can be found in tissues where no tumors are induced following administration of a carcinogen [33–36]. For example, acrylamide, which is discussed below, forms adducts in target and non-target tissues [37]. It could also be the case that epigenetic effects are required to enable neoplastic conversion resulting from some adducts [38,39].

As a result of DNA interactions, DNA-reactive carcinogens are typically genotoxic in assay systems in which appropriate bioactivation is represented [17,18,24,40,41]. Moreover, DNA-reactive carcinogens often produce tumors at multiple sites and with a short duration of exposure, even after administration of a single dose for some. This property underlies their activity in limited short-term bioassays [18].

Some DNA-reactive carcinogens have been demonstrated to exhibit no-observedadverse-effect-levels (NOAELs) for carcinogenic effects in animal models [25,31,42–46], although conflicting data have been reported. Based on the steps for tumorigenesis, it is evident that biological thresholds that may influence the likelihood of cancer progression for genotoxic carcinogens exist. Nevertheless, currently, thresholds are not generally accepted for DNA-reactive carcinogens from a risk assessment and management perspective [47]. It is acknowledged that the derivation of NOAELs can be dependent on the study design, and more research is needed in this space. It is outside the scope of this paper to discuss thresholds for carcinogens in detail; however, this topic is reviewed elsewhere [25,31,42–46].

#### *1.2. Mechanisms of Carcinogenicity of Epigenetic Carcinogens*

Epigenetic carcinogens do not chemically react with DNA [17,20,48–52]. In the target tissue(s) of carcinogenicity, MoAs of these types of carcinogens involve molecular or cellular effects, which through secondary mechanisms, can either indirectly result in modification of DNA function or cell behavior [17,48]. For example, epigenetic carcinogens can induce oxidative stress, resulting in oxidative DNA damage [53–55], leading to either neoplastic conversion or stimulation of cell proliferation, thereby facilitating neoplastic development, often from cryptogenic pre-neoplastic cells. Epigenetic carcinogens can also affect gene expression [56,57], leading to neoplastic conversion. Such effects are often specific for rodents (e.g., d-limonene). Epigenetic carcinogens can enhance carcinogenicity of DNAreactive carcinogens through interactive effects such as neoplasm promotion (e.g., butylated hydroxyanisole).

Due to their lack of direct DNA reactivity, epigenetic carcinogens, in contrast to DNA-reactive agents, are typically negative in genotoxicity assays, even in the presence of bioactivation, unless some artifact, such as extreme cytotoxicity, mediates mutagenicity. To exert their carcinogenicity, epigenetic agents often require prolonged high-level exposures. Their MoA underlies the fact that in limited bioassays they are negative for initiating activity, but may be positive for promoting activity [18].

Epigenetic carcinogens are well established to exhibit NOAELs for the cellular effect underlying their carcinogenicity in animal models [17,19], as discussed for several of the food-borne carcinogens reviewed herein. Accordingly, thresholds are generally accepted for DNA-reactive carcinogens from a risk assessment perspective [47].


**2. Linking HRM with the concept of ethics**

Before proceeding further, let us try to clarify the role of HRM in this field by defining the boundary of bribery as an unethical practice. In defining the boundary of bribery as an issue of ethics, we should consider another related concept and its counterpart—law. The basic difference between law and ethics is that if you violate the law, you will definitely be prosecuted. However, if you commit an unethical practice, you may not be prosecuted. So far, the initiatives taken by the organisations and the governments to curb the unethical practices of the people in the organisations have been proved absolutely futile. The reason is that people are not learning from the exemplary and condign punishments of the people convicted and sentenced for committing unethical activities. Law and other similar mechanisms that are in practice are like negative reinforcements and act as *preventive checks* by punishing people for committing unethical or unlawful activities. These mechanisms could not stop the repetitions of unethical activities. So, we need to find a more effective way that would curb unethical practices and would stop repetitions of unethical practices. I propose that HRM can play the first fiddle in this regard by the means of positive checks. By the term *positive check*, I propose to mean the proactive designing of HR activities, such as recruitment, job design, position description, performance management systems, and training which would change the mindset of the people in the organisation with the help of the mediating forces, such as camaraderie, collective conscience, collective voice, collective resistance, fear of ostracism, fear of obloquy, fear of public disgrace, and fear of succumbing to Dilbert principle (Adams, cited in [3], p. 8) in such a way that they would not dare to involve in the unethical practices anymore.

In this chapter, the role of HRM is examined in devising the instruments for positive checks for curbing unethical practices like bribery. This is done by examining three different cases. With the help of available secondary data, we show how appropriate HR actions could prevent the occurrence of unethical practices. In each of the cases, the following points are addressed to facilitate the understanding of the role of HRM in curbing the unethical practices:

*Bribery Practices of Three MNCs in the Host Countries: An Examination of the Issue from HRM… DOI: http://dx.doi.org/10.5772/intechopen.104810*

We conclude the chapter by identifying the particular and appropriate HR actions that could prevent the people involved from committing unethical activities. We also recommend how these HR activities can be implemented in the MNCs.


**4.6 NK, ILC cells and NKT cells**

Natural killer cells represent 30–50% of hepatic lymphocytes [63]. They differ from conventional NK cells and are closer to innate lymphoid cells (ILCs). In mouse they are closer to ILCs1, because of the expression of NK1.1 (CD161), CD69, CD49a NKp46, TRAIL. Both in mouse and human, these cells express CD49a and CD69 and secrete IFN-g and TNFa, but have weak suppressive capacity [64]. The third subset NKT cells are well presented in the liver. Different subsets are differentially presented in mice and humans, but have similar function – support immune homeostasis, control autoimmune reactions and immune responses to microbial and viral infections and cancer [65]. Of particular interest are invariant NKT and mucosal-associated invariant T cells (MAIT). They are CD3+ CD4− CD161+ Vα7.2+ cells and have robust IFN-g and granzymes B response to inflammatory signal, but limited responsiveness when stimulated directly via TCR [66, 67].

Therefore, the proper hepatic cells have dual function. On one hand they are involved in metabolic processes in the liver and on the other – they participate in immune-mediated reactions per se and by carrying out the function of a bridge between biochemical reactions and immune pathways.

Hepatic cells interact with local immune cells and thus actively participate in the establishment of a sustained immune tolerant milieu. Zheng and Tian (2019) analyzing current data, highlight the death of effector cells and the "education" of regulatory cells as key processes leading to the development of liver tolerance. Additionally, they describe a broad spectrum of baseline immune mechanisms responsible for the state of hyporesponsiveness – clonal deletion, clonal anergy, clonal deviation, T cell dysfunction/exhaustion, education etc. [68].