Session 3

Mammalian Toxicology, Session 3

Organismal regulation, feedback circuits; C 20, 21; Cell cycle controls: current models; C 3

General Comments:

While Casarett & Doull is a very good text for toxicology, there are some areas, particularly of basic physiology that are not explained as well as I might hope them to be. Endocrine and neural communication are two of these areas of minimal coverage in C& D that govern much of organismal regulation. I strongly suggest that you review some of these materials in any physiology, biology, biochemistry, cell biology, neurobiology and/or endocrinology texts that you may have from previous course-work or reading. Many illustrations, notes, and links pertinent to endocrinology can also be found at http://kcampbell.bio.umb.edu which is a public access site available on a 24/7 basis via one of the central University of Massachusetts at Boston servers. (Many of the illustrations below come from that site and may be accessed via the following links.)

Campbell Reproductive Biology Site

Endocrinology Lecture Illustrations

This page contains the illustrations that were or are going to be used in Dr. Campbell's Endocrinology Lecture Course. These illustrations, etc. may still be useful to others as they apply to many subjects used in biology.

Return to Site Directory or Endocrinology Lecture.

With respect to original journal article sources that might be used in the course or as material for your projects, the following list of journals may be helpful. Most of these can be found at the libraries of the Boston Consortium institutions (UMB, Tufts, Boston-College, Boston University, MIT,...) via library catalog searches. They are also available via many medical schools and schools of public health including Umass/Worcester. If you have Uncover access via school or work, you will be able to order specific articles you might find via PubMed or some other literature search engine. If there are items you are having difficulty finding or obtaining, please contact me and we will arrange to get them via services to which I have access. Martingale's is also a very useful site both for toxicology information and just about any other type of scientific literature or information you can imagine.

Adverse Drug Reactions and Toxicological Reviews
Annual Review of Pharmacology and Toxicology
Applied Occupational and Environmental Hygiene
Archives of Environmental Contamination and Toxicology
Archives of Toxicology
Bulletin of Environmental Contamination and Toxicology
Cell Biology and Toxicology
Chemical Research in Toxicology
Chemico-Biological Interactions
Chemotherapy
Comments on Toxicology
Critical Reviews in Toxicology
Current Advances in Toxicology
Drug and Chemical Toxicology
Ecotoxicology and Environmental Safety
Environmental Toxicology and Chemistry
Environmental Toxicology and Pharmacology
European Journal of Genetic Toxicology
Experimental and Toxicologic Pathology
Food Additives and Contaminants
Fundamental and Applied Toxicology
Human and Experimental Toxicology
Immunopharmacology and Immunotoxicology
In Vitro & Molecular Toxicology
Inhalation Toxicology
International Journal of Toxicology
Journal of Analytical Toxicology
Journal of Applied Toxicology
Journal of Biochemical and Molecular Toxicology
Journal of Environmental Pathology, Toxicology and Oncology
Journal of Toxicology - Cutaneous and Ocular Toxicology
Journal of Toxicology and Environmental Health Part A
Journal of Zoological Systematics and Evolutionary Research
Neurotoxicity Research
Pharmacology
Pharmacology & Toxicology
Regulatory Toxicology and Pharmacology
Reviews in Toxicology
Toxicologic Pathology
Toxicological & Environmental Chemistry
Toxicological Sciences
Toxicology
Toxicology & Environmental Health Part A
Toxicology and Applied Pharmacology
Toxicology and Industrial Health
Toxicology Arena
Toxicology in Vitro
Toxicology Letters
Toxicology Methods
Toxicology Modeling
Toxicon

Organismal Regulation:

Much of organismal regulation is concerned with maintaining homeostasis, both internally and with respect to responses to external stimuli. Both the central nervous system (CNS) and the endocrine system play the key roles in this network of communication. The CNS uses many of the same chemical signaling mechanisms that the endocrine system does, but tends to employ them in the more localized environment of the synaptic cleft or the neuromuscular junction. In addition the nerves of the CNS employ electrical depolarization to an extent much more clearly described in that tissue than in others to cause cellular activation. While there is no doubt that this electrochemical network, as it functions in the nervous system and the tissues activated by it, plays a key role in controlling and regulating normal function and homeostasis and because of this is of great concern when evaluating the actions of toxicants and toxins, I will limit most of my comments to chemical communications as they have been described in the endocrine system. That this is not a terrible limitation is primarily due to the fact that modern endocrinology is really the study of chemical communications within eukaryotic organisms. As such it includes much of what happens in the CNS and even touches on much of what controls prokaryotic organisms.

First, a chemical communication system consists of several parts all of which are possible targets for toxic insult. A signalling cell produces a chemical signal (usually what we call a hormone of one sort or the other) which is secreted or shed into a nondestructive carrier matrix (often plasma or intercellular fluid). Since the job of a hormone is only to convey information and since changes in needed information may change very rapidly, cells are conservative in the amount of energy they invest in producing hormone molecules. As a result, they are often produced in very small amounts of the order of 10^-9 to 10^-15 M. These small unaltered chemical signals must be sensed by target cells in the context of a complex chemical mixture that may contain closely related molecules or metabolites. This is accomplished by a chemically specific receptor that usually resides either in the cell membrane or within the cell nucleus. The receptor is allosterically altered by binding of the hormone and changes its interactions with other proteins in the cell so as to cause a sensation of the hormone molecule. This is often done via specific alterations in transducer proteins that associate with receptors and can directly generate intracellular secondary message chemicals or act on other protein and enzymatic machinery via allosteric interactions to produce an effector protein response. Since the transducer proteins often have an enzymatic activity, they amplify the original signal passed to them by the receptor-hormone complexes. This is further amplified by the effector proteins and translated into a change in cellular motion, shape, metabolism, gene transcription, or cell growth or division.

The chemical hormonal signals come in a variety of molecular types, but these can generally be classed as proteins (e.g., prolactin -- PRL, insulin, thyroid stimulating hormone/thyrotropin -- TSH, growth hormone releasing hormone/somatoliberin -- GHRH), peptides (e.g., glucagon, thyroid releasing hormone/thyroliberin -- TRH, somatostatin -- SS), amino acid derivatives (e.g., neurotransmitters like epinephrine -- E/Epi, serotonin, dopamine -- DA; thyronines/thyroid hormones like thyroxine -- T₄, or triiodothyronine -- T₃), lipids (e.g., steroids like testosterone, estradiol, or progesterone, or eicosanoids like prostaglandin E₂ or thromboxanes), or gases (e.g., NO and CO). Evidence also indicates that there is a specific membrane-bound calcium ion receptor in some cells, thus making that ion a candidate hormone. Proteins, peptides, neurotransmitters, and charged lipids like eicosanoids most often bind to receptors that are integral membrane proteins that present a binding surface at the extracellular face of target cells. Thyroid hormones, steroids, and some prostaglandins all seem to bind primarily to intracellular receptors that are often associated with DNA in the cell nucleus. These hormones are lipophilic enough to diffuse through cell membranes. Likewise gases also diffuse through cell membranes after which they appear to act via binding and allosteric alteration of enzyme activities. Notice that the various chemical properties of hormones tend to mimic the diversity of chemistries seen in toxicant chemicals including synthetics. It is not surprising then that these compounds can interact and cause alterations in many physiological systems since those systems evolved to handle a similar diversity of useful information signals.

Chemical signalling also exists in a series of distinguishable forms. It may occur between two distant cell types via hormones secreted into the blood, lymph, or intracellular fluid = endocrine signalling. It may occur between cells that are close to one another via the same fluid media = paracrine signalling. It may take place between adjacent cells (and may even involve signals and receptors that are tethered to the surfaces of the interacting cells) = juxtacrine signalling. Or it may involve cells signalling to themselves or to nearby cells of the same type = autocrine signalling.

Note also that at each level of chemical signalling modulation may take place in the normal course or development and functioning: hormone levels change over time; receptor levels change over development, time, and in response to hormone levels; transducer and effector proteins change in level and activity with development, time, and hormone levels, they may be phosphorylated or dephosphorylated, prenylated or deprenylated, and associated with intracellular activators or inhibitors depending on cellular status and condition. Moreover, although it is common for only a limited number of cells within a tissue or organism to have receptors for a particular hormone (chemical signal) it is also true that most cells in the body respond to several to many different hormones. A cell may be responding to a steroid (via a nuclear receptor and altered gene transcription) at the same time as it is responding to a neurotransmitter (via an ion channel coupled receptor) while it is also producing a peptide hormone in response to yet another protein hormone (via a cell membrane spanning receptor). It is also true that the vast majority of cells in eukaryotes do make hormones or hormone-like chemical signals during at least a portion of their lifespans. These systems are incredibly dynamic. So it is not surprising that toxicants can have so many different possible routes to generate cellular disruption. But because of this dynamism and interconnectedness, it is also quite possible to observe what appears to be a primary toxic insult in a tissue that turns out not to be the primary site of toxicant action. In fact, many of the results covered in Chapters 20 and 21 of Casarett and Doull could almost be predicted based on our current knowledge of how chemical communications operate within the body.

A bit of description about the various chemical communication networks may help illustrate this point.

Feedback Circuits:

The target cell response also frequently generates one or more chemical signals that now make this target cell into a signalling cell. The secondary hormonal signal may now impinge on other tissues to produce other cell-specific responses. Among these are frequently the original signalling cells or a set of cells that control those signalling cells. This generates a control circuit that can now operate to balance hormonal outputs in proportion to any other inputs into the cells of the control circuit as well as to the original two hormones in the control circuit described. S uch circuits are of two types: negative feedback, which is normally homeostatically balances internal chemistry and cellular functions such as gametogenesis; or postive feedback, which is usually associated with production of a physiological change of state, e.g., birth, milk let-down, ovulation. Similar controls also occur within excitable cells where protein phosphorylation and dephosphorylation often serves as a negative feedback circuit for triggering and then limiting the action of a hormonal (or electrical) stimulus or where growth factor actions produce a spiral of effects leading to cellular division.

The classical endocrine system includes several well-defined glands such as the adrenal, the testes, the ovary, the thyroid, the thymus, the pituitary, or the pancreatic islets. Those peripheral tissues that are capable of producing steroids in substantial quantities, the ovary, testis, and adrenal cortex, are under the control of specific cell types (gonadotropes, and corticotropes) in the anterior portion of the pituitary which is centrally located below the base of the brain and in a bony pocket (the sella turcica). The pituitary is connected to the base of the brain (the hypothalamus) via a well-vascularized stalk of tissue. Neurotransmitters, and several peptide and small protein hormones are produced by various portions of the hypothalamus and are secreted into the blood that circulates to the anterior pituitary. These neuroendocrine hormones (e.g., DA, TRH, SS) bind to receptors on the target cells in the anterior pituitary and stimulate or inhibit their production of protein hormone products. The pituitary protein hormones (follicle stimulating hormone -- FSH, luteinizing hormone -- LH, and adrenocorticotropic hormone/corticotropin -- ACTH) are secreted into the venous drainage of the anterior pituitary and proceed to circulate to the peripheral organs of the body. When they bind to their target cells they stimulate a variety of processes that increase production and secretion of steroids that are characteristic of the peripheral target tissue.

Vascularization of the anterior pituitary and its association with the structures of the human hypothalamus.

Control of the fasiculata and reticularis layers of the adrenal cortex by ACTH and of ACTH by the glucocortical steroids produced by the adrenal cortex (cortisol -- human, corticosterone -- rat, mouse). CRH is corticotropin releasing hormone, a small protein produced in the hypothalamus. VP is vasopressin, a nonapeptide secreted by cells of the posterior pituitary and produced by the cells of the paraventricular and supraoptic nuclei of the hypothalamus. IL-1B is a lymphokine, a protein hormone produced by lymphoid cells.

LH acts in a simple negative feedback loop in the male where it acts on the Leydig cells of the testis to stimulate testosterone production. Testosterone acts on peripheral somatic tissues and is a major player in maintaining adult sperm production. The steroid also binds to receptors in the basal hypothalamus where it suppresses production of the decapeptide hormone luteinizing hormone releasing hormone/luliberin, LHRH/GnRH. LHRH in turn stimulates the anterior pituitary gonadotrope cells to produce LH. In the female, progesterone from the temporary steroidogenic structure formed from the ovarian follicle following ovulation, the corpus luteum, CL, acts in an analogous manner to testosterone. It stimulates peripheral tissues such as the uterine lining (endometrium) to differentiate in preparation for embryonic implantation but it also acts at the hypothalamus to decrease LHRH and subsequently LH production. Since the CL is dependent on LH action for its function the negative feedback by progesterone will normally limit the lifespan of the CL and lead to its functional and structural involution. When this happens (in the absence of a fertilization) the decline in steroids associated with the CL involution leads to a rise in LH (and FSH). These initiate follicular growth in the adult ovary and stimulate production of estradiol from the granulosa cells of the follicle and the stromal cells of the ovarian tissue. As follicles grow they produce more and more estradiol which tends to act in a negative feedback manner initially similar to the actions of testosterone and progesterone with respect to LH and FSH production. Once a particular threshold is reached, however, in the later portion of the preovulatory part of the ovarian cycle, estradiol stimulates LH release by increasing the LHRH receptor numbers on the gonadotropes and, possibly, by inhibiting its own negative feedback action at the level of the hypothalamus. This positive feedback results in a spike of LH (and FSH) near the middle of the ovarian cycle that triggers the shedding of ova from mature follicles and stimulates conversion of the ovulated follicles into the next crop of CLs. Glucocortical steroids and CRH have suppressive actions on LHRH, LH, and sex steroid productivity.

The FSH control cycle is intimately tied to that of LH and is only slightly less complex. In the male testosterone suppresses LHRH, LH, and FSH production while being directly influenced only by LH. FSH stimulates the Sertoli cells of the seminiferous tubules to produce along with the proteins and metabolic products needed to help support spermatogenesis, a protein hormone, inhibin, that acts in a direct feedback manner on the gonadotropes. Primary control of FSH is due to steroid feedback with only about 20% of levels critically dependent on inhibin. In the female, granulosa cells produce inhibin in response to FSH as well as estradiol. So growing follices are actually producing both a protein and a steroid that help suppress FSH production even as they depend on FSH actions to continue their growth and development. Again, primary control of FSH is due to steroid, rather than inhibin feedback. Once the estradiol threshold necessary to convert estradiol negative feedback into positive feedback is reached, FSH levels also rise to a mid-cycle peak and then decline as progesterone takes over to suppress LHRH, LH, and FSH levels.

Control of thyroid function involves inhibition of the thyroptropes of the anterior pituitary by circulating thyroxine which acts directly as it does to promote growth and maintain metabolism on many somatic tissues in the periphery. Additionally, thyroxine can inhibit hypothalamically produced TRH. Somatostatin, a tetradecapeptide generated in the hypothalamus, can act as a secondary controller by limiting the stimulatory actions of TRH. TSH from the thyrotrope then acts selectively on the follicular epithelial cells of the thyroid to stimulate the synthesis and release of thryonines, and especially thyroxine.

Controls for production of growth hormone follow somewhat similar patterns to that for TSH and thyroxine, but prolactin is principally controlled by the suppressive effect of hypothalamic dopamine. If production of this neurotransmitter is limited in the neural circuits in the hypothalamus, PRL levels can rise in response to mammotrope/lactotrope (not luteotrope) production of the hormone. In the periphery this can affect breast tissue, immune, and reproductive tract functions. Centrally, either PRL or another controller of dopamine production, beta-endorphin, may act to affect other control circuits including LHRH production and, thereby, LH, FSH and gonadal function.

While control of insulin and glucagon from pancreatic islets are tied to regulatory circuits involving the adipose tissue protein hormone product leptin, the gastrointestinal protein hormone GHrelin, and several hypothalamic hormones including CRH, control loops also occur that seem limited to the periphery. Control of calcium and phosphate metabolism by the thyroid hormone calcitonin and the parathyroid hormone, PTH, form one such circuit. Regulation of immune function often involves ACTH, CRH, and glucocorticoids so these complex circuits may be primarily peripheral, but do retain important ties to the hypothalamus and, thus, to the CNS.

Disruption of these circuits is a primary part of diagnostic investigation in clinical endocrinology. The methods used in such efforts have been and are being used to explore toxicant insults in toxicological studies. Diagramatically, problems that disrupt the usual negative feedback circuits of homeostatic endocrine tissues (e.g., thyroid, pancreas, adrenal cortex) will tend to cause one or more of the hormones involved in that circuit to be too high (hyper-) or too low (hypo-). The balance of the hormone signals involved will often allow differentiation of where the circuit is disrupted. It seems rather obvious that toxicants that impact such circuits can also be investigated by evaluating hormonal endpoints or using the techniques used in endocrine diagnostics. Interestingly, the focus on tumor formation as a key endpoint seems to have limited the use of the hormonal parameters and endocrine techniques to explaining why such tumors form rather than how such a disruption in the internal homeostasis within an organism might have disrupted normal function up to the time of tumor formation and/or cancer production.

Cell Cycle Controls: Current Models:

Although altered metabolism is a key element of homeostasis in any organism, the ability to develop or repair tissues is dependent on the process of cell growth, mitotic division, and differentiation. And while we often think of this process as simply being regulated by availability of nonavailability of the nutrients necessary for cell division, the cellular machinery involved makes it essential that this process is highly regulated. Both intracellularly and extracellularly. This is not intuitive until several facts concerning DNA replication in eukaryotes are taken into account. First, DNA replication starts from a common point on both of the complementary strands of the molecule and it proceeds from that point in both directions at the same time while using enzymes that synthesize DNA only in one direction (5' to 3'). That involves construction of a continuous strand of deoxyribonucleotides in one direction, but a discontinuous strand of fragments (first RNA, then DNA) in the opposite direction. These fragments need to be ligated before the entire molecule is reconstructed. Failure to ligate them before the DNA fragments dissociate form their original site and reassociate, possibly incorrectly, with a sequence of similiar, but not identical composition, can result in replicative mistakes. Second, eukaryotic DNA is not only helically coiled, but it is wrapped around nucleosomal proteins, and further folded into chromosomal structures that retain substantial coiling even when they are not involved in cell division. This coiling and supercoiling forces cells to cleave their DNA while they are replicating it in order to allow it to unwind far enough to provide access to the DNA polymerase/ligase complexes. Such cleavage can also lead to mistakes unless these sites are religated soon after they are cleaved. Third, the enzymes involved in DNA replication can make mistakes. This will occur most often if there is an uneven supply of substrate nucleotides to the enzyme or if ionic composition modifies the specificity of the enzyme, e.g., if Mg⁺⁺ or Ca⁺⁺ levels vary. In addition, these cells have evolved in an environment that contains potentially damaging agents, both chemical and electromagnetic, that can alter or cleave DNA during its time in the cell. As a result, cells have produced an array of enzymes that can remove damaged segments of one strand of DNA, replace them with new nucleotides, and ligate the damaged segment back onto the parent strand. They can even repair double strand breaks to a limited degree. Thus, monitoring the condition of their own DNA and repairing it if needed is a normal cellular function. It is not, therefore, terribly surprising that similar protein and enzymatic machinery is also used during cell replication to evaluate DNA synthesis and condition prior to cell division. Nor should it be surprising that failures in these functions tend to lead to problems in daughter cells leading most often to elimination of the damaged cell, but also occasionally to the production of cells that fail to function properly and undergo a transformation often accompanied by unrestrained growth (neoplasia, tumorigenesis, carcinogenesis, malignancy). So how is mitotic cell division monitored or controlled?

Cells tend to be either in a resting or nonproliferative state, G₀ or they are actively involved in some stage of mitosis. G₁, or gap 1, phase involves cellular growth and protein synthesis. S, or synthesis, phase involves protein and metabolite synthesis in preparation for cellular DNA synthesis and finally DNA synthesis itself. G₂, or gap 2, phase involves completion of DNA synthesis and reorganization of the cellular constituents allowing for separation of chromosomes (e.g., nuclear envelop breakdown, spindle organization). M, or mitosis, phase involves the various segments of cell division: metaphase, anaphase, telophase, diakinesis. Note that prophase actually involves much of the rest of the S and G₂ phases. The "gap" phases refer to portions of the cycle during which tritiated thymidine does not incorporate into cellular DNA and cell structure cannot be used to define the cell's position in the cycle.

At the point just before S phase there is the first of two checkpoints that cells use to monitor their condition and suitability to enter mitosis. The restriction, or R, point also provides cells a means to allow extracellular input into the process of cell division. R involves the retinoblastoma, Rb, protein which acts as a brake on DNA synthesis unless growth factor or other inputs allow an override of the brake. This can occur if conditions in the organism stimulate growth factor production which then binds to cellular receptors and triggers phosphorylation of the Rb protein or if conditions within the cell provide adequate materials to allow DNA systhesis to be successful. The latter will allow accumulation of cyclin/cyclin kinase complexes that can phosphorylate Rb. This protein then dissociates from the E2F transcription factor complexes needed to promote DNA synthesis and allows DNA replication. The second checkpoint occurs just prior to commitment to mitosis, M, and involves the p53 protein. This checkpoint allows the DNA repair proteins to complete any ligation of damage or DNA-synthesis associated strand breaks. p53 binds to complexes that permit movement beyond this point and is again subject to extracellular modulation by the products of growth factor binding. Once this suppressor protein is inactivated, mitosis can proceed and daughter cells can be produced.

Should a cell fail for some reason to heed these two checkpoints, it could well pass partially broken DNA on to the daughter cells. This can lead to loss of important DNA segments or to introductions of mistakes when the DNA is repaired in the daughter cells. Since breaks often occur in areas associated with active gene transcription including genes for various portions of the signalling machinery for cell growth factors and their receptors, transducers, and effectors, mistakes may arise in these important regulatory paths. If these result in constitutive growth signalling, a cycle of inadequately regulated cell division and gradually accumulating gene loss and transformation can occur as has been found to be the case in several forms of cancer in humans including colon cancer.

More frequently problems in heeding the first checkpoint result in shunting of the cell toward programmed cell death or apoptosis. This is often triggered by p53 activation or by the presence of alternative triggering paths like Fas/Fas ligand interactions. During toxic insults the path is often activated by the intracellular production of oxidative products such as peroxide or superoxide. Many indications seem to point to the production of damage to the mitochondrial membrane and intra- as well as extracellular release of cytochrome C as a key element in activating transcription and translation of genes associated with the apoptotic cascade (e.g., caspases, Bax, Bcl) that leads to intracellular proteolysis, nucleolysis, organellar breakdown, and finally cellular dissruption by osmotic pressure and debris removal by adjacent cells. It is really only when this process fails that abnormal growths tend to occur. This may be via the production by the original cell of too many apoptosis blocker proteins, perhaps as a result of extracellular nutrient or growth factor availability. Or via dysregulation of these factors during the original omission of the checkpoints prior to daughter cell appearance.

Note that the complexity of these control paths provide ample molecular targets for toxin or toxicant disruptions of these processes. And while these will most frequently simply cause the damaged cells or their daughters to be eliminated by apoptosis and/or auto-immune surveillance by reticuloendothelial cells such as macrophages, they will occasionally result in cellular transformation. If the induction of apoptosis is widespread or is accompanied by the overt killing of cells via necrotic processes (rapid cell death usually due to sudden breach of the cell membrane or rapid loss of membrane ion gradients or osmotic gradients followed by infiltration with inflamatory cells such as lymphocytes and monocytes) tissue damage will occur. If the rate at which cell die-off occurs exceeds replication of the involved tissue or of fibroblastic (scar) replacement tissue, structural and/or functional compromise of the tissue will result.

Finally, if compromise of a regulatory tissue such as the cells of the thyroid are involved in these cell control modulations, the primary events observed will not be the result of the site of the primary lesion. Rather, they will involve the lost capacity to adequately communicate with the regulatory targets of the thyroid tissues. Heart rhythm may be disrupted by depressed thyroxine levels, unexplained weight gain may occur, sensitivity to heat and cold will be noted, CNS activity will be suppressed. All of these results will demonstrate some level of functional compromise and thus be related to a form of morbidity (which may or may not result in an early death). But none of them will immediately point to the actual primary lesion. Only by observing the constellation of effects and/or actually measuring blood hormones or the function of the thyroid gland would this insult be identified. Likewise, loss of muscle function or gastric activity may often reflect impacts on nerve tracts rather than direct impacts on the muscles or gastric tissues. Toxicological investigations must understand and take into account the normal functioning of the organism and employ all the tools available when exploring toxic mechanisms and actual toxic insults.

Assignment Work:

Journal Citations

Provide at least 3 citations gleaned from articles in the toxicology journals posted.

Calcium Controls

Describe the control circuit for parathyroid hormone, calcitonin, and cholecalciferol; how would a blocker of 1[alpha]-hydroxylase impact calcium deposition in bone?

Project Ideas

Generate a list of 3 possible case studies for possible use in the course project; associate them with an article, Website, or other document that justifies possible interest in them.