**3. Health effects of low dose bisphenol A**

Until recently, the studies on BPA mainly focused on the nuclear mechanisms of estrogen response through bind with estrogen receptors (ERs). The binding affinity of BPA to ERβ is about 10 times higher than that of ERα [52, 53]. BPA showed 10,000–100,000 weaker estrogenic potencies compared to 17β-estradiol [54]. It has been considered that BPA has relatively weak estrogenic potency due to the low binding affinity with ERs and the low estrogenic potency compared to estradiol.

However, recent studies reported a variety of molecular pathways including androgen receptor, aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor, which are associated with hormones of the endocrine and other systems in the body [34, 55]. The disrupted nuclear hormone receptors can interfere with the secretion and function of endocrine system.

#### **3.1. Low dose effects of bisphenol A**

The low dose was defined in the U.S. Environmental Protection Agency (EPA), the National Toxicology Program (NTP) assembled a group of scientists in 2001 as any biological effects occurring in the range of typical human exposures or occurring at doses lower than those typically used in traditional toxicology assessment [56]. Traditional toxicology considers that the dose makes poison. Thus, toxicological studies have been focused on identifying the concentrations at which chemicals can cause biological changes, and below that levels are not harmful to health.

According to the definition of NTP, the cutoff doses of low-dose BPA might be the range of general public exposure except for occupational exposure and the levels less than 50 mg/kg/ day of LOAEL [3, 54]. However, diethylstilbestrol (DES), which was used to prevent premature births and miscarriages of pregnant women, is one of the endocrine disruptor and is caused endocrine disrupting activity to exposed women and developing babies [57]. Thus, the safety levels of EDs may not exist.

Many experimental studies have been reported on low dose effects of BPA [1, 58, 59]. Epidemiologic studies also showed that exposure to environmental relevant levels of BPA are associated with the disorders in human [60–62]. However, there is still controversy over the low-dose effects of BPA because of the difficulty to replicate. Thus, the necessity of the reevaluation of human safety daily intake limits is raised.

Low dose is not the same as nonmonotonicity. Monotonic dose response relationship is the basic approach in traditional toxicology. In contrast to traditional toxicological approach, recent studies suggest that EDs may show the nonmonotonicity including biphasic, U- or inverted U-shape dose–response curve (**Figure 1**) [63]. The lack of monotonic dose-response relationship makes it difficult to predict the health effects at low dose using the result from high-dose endocrine disruptors.

Exposure to environmental relevant doses of BPA to pregnant mice moved the timing of vaginal opening and first estrous cyclicity up in their offspring [58]. BPA below reference dose affects the structure and functions of brain through interfering with the hormones and neuro hormone receptors [64]. It may be caused by the disruption on brain-gonads-pituitary

**Figure 1.** Examples of monotonic and non-monotonic dose response curve.

gland axis function. However, BPA exposed male and female rats showed no changes of body weight, reproductive morphology, and fertility of their female offspring [65].

**3.1. Low dose effects of bisphenol A**

130 Bisphenol A Exposure and Health Risks

the safety levels of EDs may not exist.

high-dose endocrine disruptors.

reevaluation of human safety daily intake limits is raised.

**Figure 1.** Examples of monotonic and non-monotonic dose response curve.

The low dose was defined in the U.S. Environmental Protection Agency (EPA), the National Toxicology Program (NTP) assembled a group of scientists in 2001 as any biological effects occurring in the range of typical human exposures or occurring at doses lower than those typically used in traditional toxicology assessment [56]. Traditional toxicology considers that the dose makes poison. Thus, toxicological studies have been focused on identifying the concentrations at which chemicals can cause biological changes, and below that levels are not harmful to health. According to the definition of NTP, the cutoff doses of low-dose BPA might be the range of general public exposure except for occupational exposure and the levels less than 50 mg/kg/ day of LOAEL [3, 54]. However, diethylstilbestrol (DES), which was used to prevent premature births and miscarriages of pregnant women, is one of the endocrine disruptor and is caused endocrine disrupting activity to exposed women and developing babies [57]. Thus,

Many experimental studies have been reported on low dose effects of BPA [1, 58, 59]. Epidemiologic studies also showed that exposure to environmental relevant levels of BPA are associated with the disorders in human [60–62]. However, there is still controversy over the low-dose effects of BPA because of the difficulty to replicate. Thus, the necessity of the

Low dose is not the same as nonmonotonicity. Monotonic dose response relationship is the basic approach in traditional toxicology. In contrast to traditional toxicological approach, recent studies suggest that EDs may show the nonmonotonicity including biphasic, U- or inverted U-shape dose–response curve (**Figure 1**) [63]. The lack of monotonic dose-response relationship makes it difficult to predict the health effects at low dose using the result from

Exposure to environmental relevant doses of BPA to pregnant mice moved the timing of vaginal opening and first estrous cyclicity up in their offspring [58]. BPA below reference dose affects the structure and functions of brain through interfering with the hormones and neuro hormone receptors [64]. It may be caused by the disruption on brain-gonads-pituitary In epidemiologic studies, the associations were observed between internal BPA concentrations and endocrine hormones. The BPA concentrations in the urine of men in the fertility clinic were showed inverse correlation with the estradiol:testosterone ratio [66]. Urinary BPA concentrations in human from Italy were positively associated with ERα and ERβ [67].

Recent study showed that BPA at low doses decreased estradiol level and inhibited growth of follicles isolated from wild-type and aryl hydrocarbon receptor (AHR) knock-out mice through interfering with the AHR [55]. They suggested that AHR signaling pathway might not be a major route through BPA exert its toxic effect on ovarian follicles.

The low-dose effects of BPA may associate with the genetic susceptibility, i.e., a gene-environment interaction. Transgenerational inheritance may associate with the epigenetic changes caused by low-dose BPA exposure. Without understanding the gene-environment interactions, there is a limit to understand the low dose effects. Low-dose effects of BPA should be validated through epidemiologic studies.

Exposed environmental factors during fetal or neonatal life can interact with the genome and influence the onset of diseases in their adulthood including cancer, infertility, precocious puberty, and obesity [68]. This theory is called "the developmental origins of health and disease" [69]. DES, a synthetic estrogen, is well documented that fetal exposure to DES causes the severe malformations and cancers of the reproductive tract [57].

Perinatal exposure to low-dose BPA may produce the adverse effects including brain function, reproduction, pituitary gland, and immunity (**Table 1**). The harmful effects are persisted and transferred to the fourth generation that was not directly exposed to BPA. BPA exposed fetus during their gestational period showed neoplasia and changes in mammary tissue [70].

Organ developing period as the first trimester in fetus is the critical period, which means they are extremely sensitive to low-dose effects of EDs than adult organisms. Thus, gestational exposure to EDs may induce the harmful effects on the offspring and can transfer to the subsequent generation. This process is called as "epigenetic transgenerational inheritance." The attention has been increasing to the role of epigenetic changes in the development of disease because it is considered as one of the mechanisms for explaining of low-dose effects.

When epigenetic changes are induced by EDs, those can regulate the gene expression by silencing or activating the gene. The mechanisms of regulation are classified as (1) DNA methylation, (2) histone modification, and (3) RNA-associated silencing. Because epigenetic changes do not modify the gene sequence but affect the gene expression, it may reflect the plausible association between exposure to endocrine disruptors and alteration of gene expression, which resulted into the development of disease.



**Classification** Reproductive system

Reproductive system

Reproductive system

Reproductive system

Pituitary system/Brain

Pituitary system/Brain

Pituitary system/Brain

Pituitary system/Brain

Pituitary system/Brain

Reproduction disorders

Pituitary system/Brain

Pituitary system/Brain

Pituitary system/Brain

Pregnant rats

Gavage (10, 100, 1000, 10,000

GD 10–PND 10

μg/kg bw/day)

Pregnant rats

Drinking water (0.1 mg/L BPA)

PND 0–PND 21

Pregnant rats

Pregnant rats

Drinking water (3 μg/kg bw/

GD 0–PND 21

day [estimated average dose of

exposure])

Diet (40 μg/kg bw/day)

GD 0–PND 21

Pregnant rats

Oral (5 μg/kg bw/day)

GD 1–PND 100

Pregnant rats

Pregnant rats

Oral (40 μg/kg bw/day)

SC injection (10 μg/kg bw/day)

GD 12–PND 21

GD 0–PND 21

Pregnant rats

Oral (440,400 μg/kg bw/day)

GD 6–PND 21

Pregnant rats

Gavage (2.5, 25, 2500 μg/kg

bw/day)

Pregnant rats

Pregnant rats

Drinking water (3 μg/kg bw/

day [estimated average dose of

exposure])

Gavage (2.5, 25, 260, 2700 ug/

GD 6–GD 21 (Dam gavage)/

Alters estrogen receptor

[7]

expression

PND 0–PND 21 (Pup

gavage)

GD 6–GD 21 (Dam gavage)/

Effects on anxiety or

[12]

exploratory activity

No consistent effects

Alters NCS proliferation and

[11]

differentiation

Cause anxiety like alteration

Disruption in dopamine- and

[14]

serotonin-related genes

Adverse development and

[17]

behavior effects on F1 and F2

Increase FSH, LH levels in

[6]

serum

Abnormal testis histology

Abnormal adrenal histology

[15]

Alters the basal and stress

induced activity

Alters the ERα signaling and

[16]

behavioral deficit

Alters brain development

[18]

[13]

PND 0–PND 21 (Pup

gavage)

kg bw/day)

Medaka (Oryzias

latipes)

Pregnant rats

**Animal model**

**Administration (dose)**

Gavage (50 μg/kg bw/day)

Water tank (200 ng/ml)

Lifelong, development,

neurogenesis, sex

differentiation

GD 0–PND 21

Increase LH, estradiol levels

[5]

in serumAbnormal ovary

histology

GD 6–PND 21

**Exposure duration**

**Effects (offspring)**

Reduction of semen quality

Transgenerational effects

[71]

132 Bisphenol A Exposure and Health Risks

[4]

**References**

**Table 1.** Low-dose studies of BPA in early life stage.

### **3.2. Mixed exposure**

Traditional risk assessment approaches are focused on the single chemical. Individual NOAEL does not reveal about the possible risk for the multiple exposure of EDs. Simultaneous exposure to multiple endocrine disruptors (mixed exposure) can generate combination effects even lower than their NOAEL [73, 74].

The crucial definitions for assessment of mixture exposure are classified as synergisms, antagonisms, or additivity: synergism means that the observed effects are stronger than expected; likewise, if they are weaker than expectations, there is antagonism. The combination effects are similar to the effect of individual agents are called additivism [75].

Combined effects have been reported when treated with mixture of BPA and other EDs simultaneously. *In vitro* studies, synergistic/additive effects are showed in case of simultaneous exposure of two or more chemicals [76, 77]. Perinatal exposure to low dose of BPA and paraben showed the additive effects on the downregulated semen quality in adult male offspring compared to individual exposure [4]. Mixture of BPA and other plastic-derived chemicals, despite of higher dose than environmental relevant levels, promoted epigenetic transgenerational inheritance of adult onset disease including obesity, testis, and ovary disease [78].
