Biochemical and Molecular Basis of Toxicity


Introduction

This chapter provides an overview of the fundamental principles of toxic mechanisms of injury. It is focused on biochemical, cellular, and molecular mechanisms that contribute to toxicity. Additionally, xenobiotic disposition, representing the integrated action of absorption, distribution, biotransformation, and elimination, plays a central role in the development of toxicity and is often a major determinant of the dose–response relationship for toxicity and the potential for species-specific responses (see ADME Principles in Small Molecule Drug Discovery and Development—An Industrial Perspective , Vol 1, Chap 3 and Biotherapeutic ADME and PK/PD Principles , Vol 1 , Chap 4 ).

In light of the breadth and variety of target cells, organs, and molecular pathways underlying toxic mechanisms, it is nearly impossible to adequately address the multitude of mechanisms by which toxicants elicit adverse effects. Additionally, many of the chapters that follow will address specific toxicities and pathologies relevant to a particular organ system, as well as methods and approaches for assessing the cellular, biochemical, and molecular pathways that are involved in toxic responses (see Morphologic Manifestations of Toxic Cell Injury , Vol 1, Chap 6 , Basic Approaches to Anatomic Toxicologic Pathology , Vol 1, Chap 9 , Clinical Pathology in Nonclinical Toxicity Testing , Vol 1, Chap 10 , and Toxicogenomics: A Primer for Toxicologic Pathologist s, Vol 1, Chap 15 ). Consequently, this chapter focuses on the fundamental principles that contribute broadly to toxic or pathologic effects. These include the following: (1) characteristics that determine how a toxicant is delivered to its target; (2) the major factors that determine toxic outcome; (3) factors governing whether repair and/or regeneration occur after toxic insult; and (4) the pathological states that manifest when repair mechanisms fail. Examples are provided to illustrate factors that influence the balance between the ability of an organ or cell to adapt to or repair potential toxicity.

The qualitative and quantitative features of the cellular or molecular basis of toxicity are paramount to determining the relative hazard for any toxicant. The dose–response relationship for toxicity also provides a quantitative snapshot of the continuum of effects that contribute to toxicity. Fig. 2.1 provides a general overview of the most critical determinants of toxic mechanisms and serves as the foundation for the content of this chapter.

Figure 2.1, Schematic representation of the major determinants of toxic outcomes that either limit toxicity or increase toxicity.

General Principles of Xenobiotic Disposition

The disposition of a xenobiotic is defined as the integrated action of its absorption, distribution, biotransformation, and elimination. The quantitative determination of these properties comprises the field of pharmacokinetics (or toxicokinetics) which is discussed in detail in ADME Principles in Small Molecule Drug Discovery and Development—An Industrial Perspective , Vol 1, Chap 3, Biotherapeutic ADME and PK/PD Principle s, Vol 1, Chap 4 , and Principles of Pharmacodynamics and Toxicodynamics , Vol 1, Chap 5 ). Collectively, the disposition and kinetics of a chemical ultimately determine its concentration at a target site for toxicity and contribute to whether adverse effects will occur.

General Properties of Absorption

A comprehensive overview of basic dispositional parameters and attributes (absorption, distribution, and excretion) is not provided here. Additional relevant information covering absorption, distribution, and excretion can be found in the literature ( , ( Chapter 5 ).

Biological Membranes

Cell membranes are comprised of a phospholipid bilayer wherein the polar head groups of the lipids are oriented toward the outer and inner surfaces of the membrane and the lipid tails are oriented inward forming a hydrophobic inner space. Cell membranes are typically 7–9 nm thick, and phosphatidylcholine and phosphatidylethanolamine are the primary phospholipids in the outer and inner leaflets, respectively. They also contain a variety of transmembrane proteins that function as receptors for many endogenous ligands, form pores or ion channels or function in the transport of endogenous and exogenous compounds into and out of cells (discussed below). The processes involved in the passage of compounds across membranes include those that require no energy (e.g., direct passage through pores, filtration, or simple diffusion). Additionally, there are numerous active transport processes, defined as those which require energy utilization to move solutes across membranes and against a concentration gradient.

Simple Diffusion

Diffusion occurs down a concentration gradient, with molecules moving from regions of higher concentration to lower concentration (Fick's law). Large hydrophobic molecules diffuse across the lipid domain of membranes. In contrast, smaller water-soluble molecules can pass through aqueous pores in a process referred to as paracellular diffusion. This is the case for ethanol, and this attribute enables absorption readily across the gastrointestinal (GI) tract and distribution rapidly throughout the body.

Two principal factors that govern the rate of transport across membranes for large organic molecules are lipid solubility and the degree to which a compound is in its nonionized form at physiological pH. Lipid solubility is frequently expressed as the octanol–water partition coefficient (or log P), where a very lipid-soluble molecule has a positive log P. For example, the highly lipophilic compound, 2,3,7,8-tetrachlorodibenzodioxin (TCDD) has a log P of 7.05, whereas the metal salt, lead acetate, has negative log P (−0.63). Ionization is determined by the Henderson–Hasselbalch equation which defines the relationship between the pH and the pK (i.e., the pH at which a weak organic acid or base is 50% ionized). Only the nonionized form of a compound is available for diffusion across membranes. In this manner, a weak base, such as aniline (pK = 5), is 50% ionized at pH 5, but is essentially in its nonionized form at pH 7. Thus, physiological pH favors the absorbance of weak bases more so than weak acids.

Filtration

Filtration is the bulk movement of water across a porous membrane, and any solute that is small enough to pass through membrane pores will flow with it. Overall, filtration is governed by hydrostatic and osmotic pressures along with the pores that allow small molecules to pass through them. Pore sizes are typically 2–7 Ǻ, and as pressure rises on one side of the membrane, small molecules are forced through the pores. The renal glomerulus is a major site of filtration, and its pores are typically in range of 70–80 Ǻ, allowing numerous solutes and some low molecular weight proteins to be filtered. In some endothelial beds, there are larger pores that form interendothelial gaps to allow larger molecules to move from the plasma to extracellular space. In contrast, such pores are absent in the brain where the blood–brain barrier (BBB) is formed by tight junctions between cells.

Specialized Transport: Active Transport and Facilitated Diffusion ( )

The sequencing of the human genome revealed that there are at least 500 genes likely to function in membrane transport. These systems regulate uptake and efflux of compounds and contribute to the homeostasis of endogenous compounds and xenobiotics, including drug responses and the potential for adverse effects. There are active transporters that utilize energy (typically ATP) to move chemicals against a concentration gradient. They demonstrate some substrate selectivity, with saturation at high concentrations and inhibition by compounds that compete for transport. There is growing evidence that genetic polymorphisms and species differences in transporter function and regulation contribute to interindividual and interspecies differences in toxicity, respectively. Furthermore, transporter activity can influence toxic responses because of saturation or competition for transport which is often the case in drug–drug interactions.

The first ATP-dependent transporter identified to play a role in xenobiotic disposition was a phosphoglycoprotein identified in tumor cells that developed resistance to chemotherapy. The protein was called P-glycoprotein (P-gp) or multidrug resistance protein (MDR1), and subsequently, two forms (Mdr1a and Mdr1b) have been identified in rodents. There are now several transporter families identified that contribute to the disposition of xenobiotics and influence toxicant exposure and outcome. In addition to the MDR family, these include the following: multidrug resistance proteins (MRPs), breast cancer resistance protein (BCRP), bile salt export pump (BSEP), multidrug and toxin extrusion transporters (MATEs), organic anion transporting polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs), and peptide transporters (PEPTs). A summary of these gene families is provided in Table 2.1 . Additionally, although not summarized here, there is increased interest in these transporters in veterinary practice, with an accumulating body of information on the expression and function of many transport proteins in livestock and animals of veterinary interest ( ). A full review of the function of xenobiotic transporters is beyond the scope of this chapter, but examples of how these proteins influence exposure to toxicants or contribute to mechanisms of toxicity will be discussed throughout.

Table 2.1
Summary of Major Transporters That Contribute to Xenobiotic Disposition a , b
Name Gene family Function Common name Tissue expression
Multidrug resistance protein; P-glycoprotein Abcb1 Efflux pump Mdr1a, 1b Liver, kidney, brain, small intestine, and skin
Bile salt export pump Abcb11 Efflux pump for bile salts Bsep Liver
Multidrug resistance protein (MRP) Abcc Efflux pumps; apical and/or basolateral Mrp1 Choroid plexus and skin
Mrp2 Liver, kidney, brain, and small intestine
Mrp3 Liver, small intestine, and skin
Mrp4 Liver, kidney, brain, choroid plexus, and skin
Mrp5 Brain and skin
Mrp6 Liver
Breast cancer resistance protein (BCRP) Abcg2 Efflux pump on bile canaliculus Bcrp Liver, kidney, brain, and placenta
Organic anion transporting polypeptide (OATP) Slco Influx pump; organic anions substrates Oatp1a1 Liver, kidney, and choroid plexus
Oatp1a4 Liver, kidney choroid plexus, and brain
Oatp1b2 Liver
Oatp 2a1 Kidney
Organic anion transporter (OAT) Slc22 Uptake of organic anions Oat1 Kidney
Oat2 Liver and kidney
Oat 3 Kidney and brain
Organic cation transporter (OCT) Slc22 Uptake of organic cations Oct1 Liver, kidney jejunum, testis, and skin
Oct2 Kidney, lung, and choroid plexus
Oct 3 Testis and skin
Organic cation/carnitine transporter Slc22 Carnitine transport Octn1 Kidney and skin
Carnitine transport Octn2 Kidney, small intestine, and skin
Multidrug and toxin extrusion transporter (MATE) Slc47 Efflux pump for cations Mate1 Kidney and liver
H + /cation antiporter MATE2K Human kidney

a Genes listed represent major transporters in rats involved in xenobiotic disposition (except where noted for MATE2K). Several transporter families including those that contribute to nucleoside or peptide transport are not included but are discussed in the text.

b Reference: Klaassen CD, Aleksunes LM: Xenobiotic, bile acid, and cholesterol transporters: function and regulation, Pharmacol Rev 62, 1–96, 2010.

The process of facilitated diffusion is also a carrier-mediated process, but it does not require ATP consumption. In facilitated diffusion, a substrate moves with a concentration gradient, and carrier proteins, typically integral membrane proteins, are responsible for the passage of molecules or ions across the membrane. Classically, the transport of glucose occurs by facilitated diffusion. Additionally, the OCT family, which is highly abundant in liver and kidney, moves cations by a facilitated process.

In addition to transporters that contribute to the disposition of xenobiotics, numerous other families are specifically involved in the distribution of important endogenous compounds. These include the glucose transporters described above (gene family SLC5A ), nucleoside transporters (gene family SLC29A ), and other transporter families involved predominantly in uptake of basic or neutral amino acids, including neurotransmitters, and transporters involved in the distribution of essential elements such as calcium, iron, and copper. The energy requirements of these transporters vary across families, as some, but not all, require ATP. More importantly, although such transporters are associated with key endogenous nutrients, they can influence exposure to toxicants. For example, lead is a substrate for the facilitated transporters involved in calcium and iron uptake. Furthermore, 1-methyl-4-phenylpyridinium selectively targets dopaminergic neurons and leads to Parkinson-like neurotoxicity because its uptake is regulated by the dopamine transporter (SLCA3).

Routes of Absorption

Absorption from the Gastrointestinal Tract

Absorption can occur all along the GI tract, but as noted earlier, the nonionized fraction is most readily absorbed. Due to the change in pH throughout the GI tract, some weak acids may be absorbed in the acidic pH of the stomach, whereas most weak bases are not absorbed until reaching the more neutral environment of the small intestine. The mammalian GI tract has numerous specialized transport systems for the absorption of nutrients and electrolytes. For example, the absorption of iron depends on the levels of the nutrient in the body and takes place in two steps: Iron first enters the mucosal cells and then moves into the blood. Uptake is relatively rapid, whereas entry into the circulation is slower, leading to accumulation within the mucosal cells as a protein–iron complex (ferritin). When the concentration of iron in blood decreases, it can be liberated from the mucosal stores of ferritin and transported into the blood. Cobalt and manganese (Mn) also utilize and compete with the iron in this transport system, therefore exogenous exposures to the heavy metals can result in changes in iron metabolism and toxicity.

Numerous xenobiotic transporters are expressed in the GI tract, where they function to increase or decrease absorption of these compounds. Influx transporters are predominantly localized on the apical brush border membranes of enterocytes and increase uptake from the lumen into the cells. These include the OATPs, OCTs, and peptide transporters (Pept1). The primary active efflux transporters such as P-gp, Mrp2, and BCRP are also expressed on enterocyte brush border membranes, where they function to excrete their substrates into the lumen. Substrates for efflux transporters show reduced absorption, an action that may limit toxicity. However, these transporters can also limit the oral absorption of drugs. For example, the immunosuppressive drug cyclosporine and chemotherapeutics such as paclitaxel and vincristine are poorly absorbed after oral administration because they are good substrates for P-gp.

The expression patterns of these transporters throughout the duodenum, jejunum, ileum, and colon are not equivalent. P-gp (MDR1) expression in the intestine increases from the duodenum to colon, whereas Mrp2 expression is highest in the duodenum and decreases to undetectable levels in the terminal ileum and colon. BCRP is distributed uniformly throughout the small intestine and colon.

Particles can also be absorbed by the GI epithelium. Particle size is the primary determinant of absorption, and smaller particles are more likely to be absorbed. Absorption of nanoparticles in rats is as high as 30% for particles that are 50 nm in diameter, and nonionized particles are better absorbed than ionized ones. Absorption into gut-associated lymphoid tissue (e.g., Peyer's patches) and the mesenteric lymph supply is key to systemic absorption. The amount of a chemical that enters the systemic circulation after oral administration depends on the amount absorbed into the cells of the GI tract, the action of transporters on uptake or efflux from the cells, and the potential for biotransformation. An important concept in this regard is presystemic elimination, referred to as a first-pass effect, which is the potential for removal of chemicals before entrance into the systemic circulation. Chemicals that have a high first-pass effect will appear to have lower absorption because they are eliminated as quickly as they are absorbed. Metabolizing enzymes or efflux transporters in the GI tract also contribute to the first-pass effect by limiting the absorption of the chemical to the liver or systemic circulation. A classic example is grapefruit juice that contains a naturally occurring flavonoid, naringin, which directly inhibits P-gp (and cytochrome P450 (CYP) 3A activity, discussed below) ( ). As such, drinking grapefruit juice will increase absorption of some xenobiotics because it inhibits their intestinal efflux and metabolism.

Absorption from the Respiratory Tract

Agents that are absorbed by the lungs include gases, vapors of volatile liquids, aerosols, and particulates. The absorption of inhaled gases takes place mainly in the lungs, but they first must pass through the nose where some molecules are retained if they react with cell surface components. Although such actions may reduce systemic exposure or protect the lungs from damage, they also increase the potential for the nose to be adversely affected. Such is the case with formaldehyde and vinyl acetate which cause tumors of the nasal turbinates in rats ( ). However, with formaldehyde species, differences in respiratory function, including the ability to inhale through both the nose and mouth, contribute to species differences in carcinogenic outcome. Absorption of gases in the lungs is determined primarily by the respiration rate. It differs from GI absorption because most ionized molecules have low volatility and do not achieve significant concentrations in ambient air. Overall, any chemical absorbed by the lungs is removed rapidly by the blood, which moves very quickly through the extensive capillary network.

Absorption of aerosols and particles is determined by the size and water solubility of any chemical in the aerosol. In general, the smaller the particle, the further into the respiratory tree the particle will deposit, resulting in efficient absorption. Particles ≥5 μm usually are deposited in the nasopharyngeal region, whereas those with diameters of approximately 2.5 μm are deposited mainly in the tracheobronchiolar regions, from which they may be cleared by retrograde movement of the mucus layer in the ciliated portions of the respiratory tract (mucociliary escalator). Particles 1 μm and smaller travel to the alveolar sacs where they can be readily absorbed in alveoli. Nanoparticles (less than 0.1 μm in diameter) are most likely to deposit in the alveolar region where they may be absorbed into the blood or cleared through lymphatics after being scavenged by alveolar macrophages ( ; ). For more information, see Nanoparticulates , Vol 2, Chap 31.

Following exposure and deposition, the removal of some particles from the alveoli is relatively inefficient, and the particles may remain indefinitely (e.g., coal dust, asbestos fibers, and carbon nanotubes). The deposition of these types of particles can reduce mucociliary clearance and/or they may be engulfed by alveolar macrophages where they can be retained for years. The long-term sequelae of such deposition is associated with a variety of chronic lung diseases.

Absorption Through the Skin

As the largest organ in the body, skin comes into contact with many chemicals, but exposure is usually limited by its relatively impermeable nature. The stratum corneum is the single most important barrier to preventing fluid loss from the body while also serving to prevent the absorption of xenobiotics. Chemicals move across the stratum corneum by passive diffusion, and in general, diffusion is proportional to their level of lipid solubility and inversely related to molecular weight. Once absorbed across the stratum corneum, the vascular network within the dermis allows these absorbed compounds to enter the body. The skin, particularly keratinocytes, expresses numerous drug metabolizing enzymes and xenobiotic transporters (e.g., Mrp 1, 3, 4, 5, and 6) that can aid or protect against xenobiotic absorption.

Dermal absorption varies widely across species. In general, rodent skin allows greater uptake than human skin, whereas the cutaneous permeability characteristics of guinea pigs, pigs, and monkeys are similar to those in humans. Species differences in dermal absorption of xenobiotics result from several anatomic, physiologic, and biochemical factors. Importantly, the stratum corneum is much thicker in humans than in most laboratory animals. However, the thinner stratum corneum in animals is often compensated for by a relatively thick hair cover, diminishing direct contact of the skin with a xenobiotic. In addition, expression of drug metabolizing enzymes and xenobiotic transporters may contribute to differences in dermal absorption of toxicants across species ( ).

General Principles of Distribution

Once absorbed, a toxicant distributes throughout the body, and this process usually occurs rapidly. The rate of distribution to tissues is determined primarily by blood flow and the rate of diffusion out of capillary beds into a particular organ. The liver is often a major organ for initial distribution given the relatively high blood flow ( Table 2.2 ) and its role in metabolism (discussed below). The final distribution depends on the affinity of a xenobiotic for various tissues, and this factor often determines the target organs of toxicity.

Table 2.2
Estimates of Blood Flow in Liver and Kidney Across Species
Reference: Lipscomb JC, Poet TS.: In vitro measurements of metabolism for application in pharmacokinetic modeling, Pharmacol. Ther 118, 82–103, 2008.
Estimated blood flow (mL/min/kg)
Species Hepatic Renal Total
Mouse 90 14 400
Rat 70 5 300
Dog 40 6 120
Monkey a 44 2 220
Human 20 1.8 80

a Monkey denotes cynomolgus monkey; all results are estimates (numerous literature sources) for comparative purposes.

Volume of Distribution

The volume of distribution (Vd) is used to quantify the distribution of a xenobiotic throughout the body. It is defined as the volume (L) in which the amount of compound would need to be uniformly dissolved in order to produce the observed blood concentration. If a chemical is only in the plasma compartment and not distributed into tissues, it would have a higher plasma concentration and a low Vd. In contrast, if a compound distributes throughout the body, it exhibits much lower concentrations in the blood and a high Vd. Some toxicants selectively accumulate in certain parts of the body as a result of protein binding, active transport, or high solubility in fat.

Storage of Toxicants in Tissues

The most important storage sites for toxicants are plasma, due to protein binding, and tissue depots. The site of disposition and the target organ of a chemical or toxicant can be the same, but toxicants can also accumulate in organs that differ from the site of toxicity. The lung toxicant paraquat or renal toxicants such as adefovir or cis-platinum show selective accumulation in the target organ of toxicity. However, lead is stored in bone but demonstrates toxicity in soft tissues.

Serum albumin, the concentration of which is about 500–600 μM in all species, is a major binding site for compounds in plasma. A second relevant serum protein is α 1 -acid glycoprotein that is present in all species but at a much lower concentration than albumin. The degree of binding of chemicals to plasma proteins can determine toxicity because only the unbound fraction can enter tissues. Therefore, a compound that is highly bound to plasma protein may not show toxicity when compared to one that is less extensively bound to plasma proteins. Ironically, a high degree of protein binding can increase the risk of adverse effects resulting from interactions with other highly bound compounds. In particular, severe reactions can occur if a toxicant with a high degree of protein binding is displaced from plasma proteins by another agent, increasing the free fraction of the compound in plasma. For example, if a strongly bound sulfonamide is given concurrently with an antidiabetic drug, the sulfonamide may displace the antidiabetic drug and induce a hypoglycemic coma. Similarly, interactions resulting from displacement of warfarin can lead to inappropriate blood clotting and deleterious effects. Xenobiotics can also compete with and displace endogenous compounds that are bound to plasma proteins.

The liver and kidney have a high capacity for binding many chemicals. These two organs probably concentrate more toxicants than all the other organs combined, and in most cases, active transport or binding to tissue components is likely to be involved. The heavy metal binding protein metallothionein (MT) sequesters both essential and toxic metals, including zinc (Zn) and cadmium (Cd), with high affinities in the kidney and liver. In liver, Cd is sequestered and bound to MT, preventing its toxicity despite high hepatic levels. In contrast, glomerular filtration of the Cd–MT complex can result in renal toxicity due to proximal tubule damage. For more information, see Heavy Metals , Vol 2 , Chap 27].

Another protein that sequesters toxicants in the kidney is α2u-globulin. This protein, which is synthesized in large quantities only in male rats, binds to a diverse array of xenobiotics, including metabolites of d -limonene (a major constituent of orange juice) and 2,4,4-trimethylpentane (found in unleaded gasoline). The chemical–α2u-globulin complex is taken up by the kidney, where it accumulates within the lysosomal compartment and damages the proximal tubule epithelial cells. Ultimately, the accumulation of this complex in the kidney, termed α2u-globulin or hyaline droplet nephropathy, is responsible for male rat-specific nephrotoxicity and carcinogenicity.

Adipose tissue is a storage site for chemicals that have a high lipid/water partition coefficient. This is an issue for environmental bioaccumulation and can contribute to low-grade chronic exposure to persistent chemicals. Adipose storage is a critical factor in the toxicity of lipophilic pesticides such as chlordane and dichloro-diphenyl-trichloroethane (DDT), along with the large class of polychlorinated and polybrominated biphenyls, dioxins, and furans (see Agro/Bulk Chemicals , Vol 2 , Chap 12 ] and Environmental Toxicologic Pathology , Vol 2 , Chap 18]). The potential for these compounds to produce toxicity, including carcinogenic, developmental, and endocrine effects, is related to their accumulation and storage in adipose tissue. Although storage in adipose tissue may reduce the amount that reaches target organs, there is a risk that mobilization of lipids from storage sites will increase the concentration of a chemical in blood and the target organ(s) of toxicity ( ).

Bone is an important depot for compounds such as fluoride, lead, and strontium, all of which may be incorporated and stored in the bone matrix. For example, 90% of lead in the body is eventually found in the skeleton, although it is not toxic to bone itself. In contrast, deposition in bone is an important determinant of the toxicity of fluoride and radioactive strontium (see Bone and Joints , Vol 3 , Chap 4).

Barriers Affecting Distribution

Blood–Brain Barrier

Several organs, most notably the brain, have barriers that restrict entry to toxicants. The BBB is formed primarily by endothelial cells with tight junctions between adjacent cells that prevent diffusion of polar compounds through paracellular pathways. Four ATP-dependent transporters also function as part of the BBB including P-gp, Mrp2, Mrp4, and BCRP. These efflux transporters are located on the luminal plasma membrane and prevent distribution to the brain by pumping xenobiotics absorbed into the capillary endothelial cells back out into the blood [see Nervous System , Vol 3 , Chap 9]).

Genetically modified mice illustrate the importance of transport processes to the maintenance of the BBB and restriction of compounds from the brain. For example, compounds that are substrates for P-gp achieve much higher brain levels in P-gp null (Mdr1a –/– /Mdr1b –/– ) mice relative to wild-type mice. Seminal studies demonstrated that compounds like ivermectin and vinblastine accumulated in the brains of P-gp null mice leading to marked neurotoxicity and increased lethality ( ; ).

Gut Barrier

The gut is a composite of both physical and functional barriers that protect organisms from pathogens while remaining selectively permeable for the absorption of nutrients, electrolytes, and water. The barrier is composed of gut microbiota, mucus, epithelial cells, and gut-associated lymphoid tissue or gut-associated immune cells. Intestinal permeability is described as two major pathways, namely transepithelial/transcellular and paracellular. As discussed previously, major transporters expressed in enterocytes throughout the GI tract, such as P-gp and Mrp2, can alter bioavailability of xenobiotics. A widely used cellular system to assess intestinal permeability of xenobiotics is the Caco-2 cell line, an immortalized line of human colorectal adenocarcinoma cells ( ; ). This intestinal system mimics the epithelial barrier by creating an apical brush boarder, tight junctions, and expressing enzymes and transporters within the epithelial monolayer. As in the BBB, passage of molecules through the space between intestinal epithelial cells is controlled by tight junction proteins that are highly regulated, traverse the intestines, and disruption can contribute to disease.

Intestinal barrier dysfunction is often secondary to immune-mediated mechanisms wherein cytokines, released locally as a result of conditions such as inflammatory bowel disease, food allergies, celiac disease, and diabetes, increase permeability and alter absorption of nutrients and drugs. Ethanol metabolism from chronic alcohol consumption has been associated with increased intestinal permeability and endotoxin translocation due to high levels of the metabolite acetaldehyde in the intestine. Both cytokine (e.g., interferon gamma or tissue necrosis factor alpha (TNF-α)) and acetaldehyde treatment disrupt the integrity of tight junctions increasing permeability of the Caco-2 monolayer in vitro. Lastly, xenobiotics such as nonsteroidal antiinflammatory drugs or immunotherapy checkpoint inhibitors demonstrate a multistage process in GI toxicity that alters the integrity of the gut barrier ( ). For example, adverse intestinal side effects have been observed with grapiprant treatment in dogs and anticytotoxic T-lymphocyte–associated protein-4 (CTLA-4) therapy in humans ( ; ).

Placental Barrier

The placenta is a specialized structure that serves to nourish and protect the developing fetus. Although its organization differs markedly across species, the major cellular elements are the syncytiotrophoblast and cytotrophoblast layers (see Embryo , f etus and placenta, Vol 4 , Chap 11). Xenobiotic transporters are differentially expressed in various cells of the placental unit and contribute to the barrier function that restricts distribution of toxicants to the fetus. Importantly, in most species, BCRP expression is highest in the placenta relative to any other tissue, where it plays a pivotal role in protecting the fetus from exposure to toxicants. Other transporters, namely P-gp and Mrp2, are expressed along with BCRP on the apical border of the syncytiotrophoblast, whereas Mrp1 is localized to the basolateral membranes of these cells and the fetal capillary endothelial cells ( ).

Despite the presence of biotransformation systems and xenobiotic transporters designed to nourish and protect the fetus, the transfer of xenobiotics across the placenta can still occur. One important consequence of placental transfer is that of transplacental carcinogenesis. In this case exposing the mother during gestation increases the likelihood of tumor development in the offspring later in life (see Carcinogenesis: Mechanisms and Evaluation , Vol 1 , Chap 8 ) . The most well-known transplacental carcinogen in humans is diethylstilbestrol, but other compounds such as the antiviral drug zidovudine and inorganic arsenic induce tumors in mice when exposed to these chemicals only during gestation ( ; ; ).

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