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CRISPR/HDR Platform Allows for the Production of Monoclonal Antibodies with the Constant Region of Choice
By Sharon Yang, Cell Biology, ‘20
Author’s Note: I first came across an article talking about this new innovation on Science X. Having worked with hybridomas and antibodies through various internships, I was deeply intrigued by this discovery and secured an original paper to learn more about its potential applications. Because of the revolutionizing usage of antibodies in the medical field, it is vital to understand how this finding will facilitate antibody-based therapies in clinical research.
Introduction
Since the discovery of antibodies and their applications in therapeutics, many diseases once deemed incurable now have a treatment, if not a cure. Antibodies are proteins that recognize and bind to specific antigens (proteins that are considered “foreign” to the body). The immune system recognizes this antibody-antigen complex and removes the foreign substance from the body. Monoclonal antibodies (mAbs) are specific for one type of antigen and are produced using hybridomas, immortal cell lines that secrete only one type of antibody. The specificity of a mAb is determined by its antigen binding variable region. Though the variable region is of critical importance, the constant region (also known as the Fc region) is also essential to the therapeutic efficacy of mAbs. The Fc region has many different variants, called isotypes. Each isotype has its own unique function in making the immune system respond in different ways. After an antibody binds to an antigen by its variable region, the Fc region of the antibody elicits a response from the immune system, which serves as the basis for antibody-based therapeutics.
A recent study conducted in the summer of 2019 by Schoot and colleagues demonstrates how the use of genetic engineering on hybridomas can modify the Fc region of mAbs to that of a different species, isotype, or format. This new versatile platform grants ease of production of monoclonal antibodies that have different constant regions but retain the same variable regions.
The research team utilized a one-step clustered regularly interspaced short palindromic repeat (CRISPR)/homology-directed repair (HDR) technique to create a recombinant hybridoma that secretes a mAb in the Fc format of choice — a highly attractive alternative to the conventional recombinant production methods that were often time-consuming, challenging, and expensive.
As the team emphasizes, “[CRISPR/HDR] is a simple alternative approach requiring a single electroporation step to obtain an unlimited source of target antibody in the isotype format of choice” (1). Through using CRISPR/HDR, the team was able to seamlessly generate monovalent Fab’ fragments and a panel of different isotypes for the same monoclonal antibody.
CRISPR/Cas9 and Homology-Directed Repair
In their genetic engineering method, the researchers took advantage of an ancient bacterial immunity mechanism: the CRISPR/Cas9 system. When a bacteria is invaded by a virus, the bacteria stores snippets of viral DNA and creates segments of DNA called CRISPR arrays. When a virus with the same DNA segment attacks again, the bacteria creates RNA from the CRISPR arrays to target the virus; the RNA is called the guide RNA. The nuclease protein Cas9 is used to cut the DNA apart at a very specific site determined by the guide RNA, disabling the virus. CRISPR/Cas9 works in a similar fashion in the lab. Scientists create a guide RNA that binds to Cas9, which then targets a specific site on the DNA to be cut (2).
When CRISPR/Cas9 cuts DNA, it induces a double-strand break (DSB). Homology-directed repair (HDR) occurs when the intact donor strand contains high sequence homology to the damaged DNA strand. Through HDR, scientists can integrate a sequence or gene of their liking into the genome, which Schoot and colleagues perform in their study (3).
The Generation of Fab’ Fragments
The fragment antigen-binding (Fab’) is a region on the antibody that binds to the antigen. It consists of a single heavy chain and light chain. To create a Fab’ fragment-secreting hybridoma using CRISPR/HDR, the team selected NLDC-145, a hybridoma clone that secretes mAbs of rat IgG2a (rIgG2a) isotype. The antigen of rIgG2a is DEC205, an endocytic receptor found on immune cells. The team electroporated NLDC-145 cells with Cas9 and an appropriate guide RNA to induce double-strand breaks at the hinge region; to repair the double-strand break, they designed an HDR Fab’ donor construct for homology-directed repair. The HDR Fab’ donor construct also inserts specific tags onto the protein, allowing for easy purification of the Fab’ fragment.
To test secretion of the Fab’ fragment, they stained JAWSII, a DEC205-expressing cell line, with the supernatants of NLDC-145 clones that had undergone CRISPR/HDR. Flow cytometry assays showed that a large portion of Fab’-secreting hybridomas were successfully created. Further assays showed that the secreted Fab’ fragments retained their binding capabilities. It is worth noting that the researchers also used the same strategy to convert other hybridoma lines to become recombinant, Fab’-producing lines, with similar success; this demonstrates that this engineering technique is flexible and not just limited to one cell line (1).
The Generation of Isotype Panels
In a similar manner to creating monoclonal Fab’-generating hybridomas, the team also used the one-step CRISPR/HDR technique to create hybridomas capable of producing a wide array of isotype variants for the same mAb. This time, the cell line subject was hybridoma MIH5, which secretes monoclonal rIgG2a that targets mouse PD-L1, an immune checkpoint protein. The goal was to make clones of MIH5 to each produce one isotype of the chimeric (having both rat and mouse-related parts) monoclonal antibodies: mIgG1, mIgG2a, mIgG2b, mIgG3, mIgA, and a mutant form of mIgG2a (mIgG2asilent).
MIH5 cells were cotransfected (introduced with DNA) with a Cas9 vector containing the appropriate guide RNA and a construct from a panel of isotype HDR donor constructs (each isotype had its own unique HDR donor construct). Following knock-in integration, flow cytometry analysis showed that the engineered chimeric mAbs were successfully secreted. Thus, the creation of recombinant hybridomas for a panel of isotypes was successfully engineered (1). This invention allows for the creation of monoclonal antibodies with different Fc regions, providing researchers an easy way to “customize” their antibodies to elicit a specific response from the immune system. Researchers may choose which isotype variant they want on their antibody, which is fully dependent on their target (antigen) of interest and how the immune system behaves towards it. This has vast potential in antibody-based therapeutics, in that this system can be used for the optimization of potential drugs to become more potent and dynamic.
Biochemical Applications
To test the functional capability of isotype-switched mAbs, Schoot and colleagues tested the antibodies’ capability to induce an important immune mechanism: antibody-dependent cellular cytotoxicity (ADCC). In order to test ADCC in vitro, mouse colon adenocarcinoma cells were labeled with chromium-51, and then taken in by MIH5 Fc isotype variants. After adding whole blood, they measured chromium-51 release. On the other hand, B cell depletion by MIH5 Fc variants was used to measure ADCC in in vivo experiments. Analyses of these studies show that chimeric mAbs created by CRISPR/HDR hybridomas have the same biochemical and immune effector characteristics as their recombinant and naturally occurring counterparts (1). Something to highlight is that instead of treading through the laborious process of producing recombinant antibodies in the conventional way (often consisting of multiple rounds of optimized sequencing, cloning, transfection), this one-step mechanism grants smooth and rapid generation of recombinant antibodies that perform their expected functions (1).
Conclusion
The ability to create monoclonal antibodies with the freedom to choose what goes on their constant regions possess many applications in the vast field of medicine and engineering. Being able to construct a very specific monoclonal antibody (the engineering element) that stimulates the immune system in a certain, beneficial way (the medical component) intertwines the two fields together to propel us closer towards treating diseases more efficiently and effectively. This system also represents an optimized version of recombinant engineering, which saves valuable time and funds that can be used towards conducting further studies. A simple, yet powerful and flexible approach, this versatile CRISPR/HDR platform aims to facilitate antibody engineering and research for the scientific community, and is accelerating the rate at which new clinical trials can be performed.
References
- Schoot, J. M. V. D. et al. Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering. Science Advances 5, (2019).
- Ran, F Ann et al. “Genome engineering using the CRISPR-Cas9 system.” Nature protocols vol. 8,11 (2013): 2281-2308. doi:10.1038/nprot.2013.143
- Cortez, Chari. “CRISPR 101: Homology Directed Repair.” Addgene Blog, Addgene, 12 Mar. 2015, blog.addgene.org/crispr-101-homology-directed-repair.
Robot-Assisted Surgeries
By Neha Madugala, Cognitive Science, ‘22
Author’s Note:
I came across an article detailing the future of surgery. What initially seems like science fiction may be becoming a reality as more and more surgeries are being administered by robots. Through my research, however, I found that robot-assisted surgeries may have the initial appeal of lowering human error, but there are still various issues that must be resolved before they can fully take over in the surgical room.
Robot-assisted surgeries boast the potential of shorter recovery time, less pain and blood, and fewer scars and infections. They have been on the market for a little less than twenty years, and have been used in cancer procedures for about the past fifteen years. While the FDA has approved these devices for other procedures, robot-assisted surgeries have not officially been approved for cancer treatments. Regardless, surgeons have been and continue to perform robot-assisted surgery for cancer-related procedures due to their benefits and increased efficiency.
Robot-assisted surgeries mainly contrast from traditional surgeries because they can be performed through small cuts in the patient’s body. As a result, they are minimally invasive. There are three robotic arms, allowing for multiple angles for improved accuracy, which perform the incisions. According to The New York Times, the robotic arms are controlled by a computer and software that replicates the operating surgeon’s movements. This occurs as the operating surgeon performs the movements while looking at a magnified and high-definition screen of the surgical site captured by a camera attached to the robot. While the device requires limited retraining for surgeons, as of now, there is only one company actually offering this device. Interestingly, the device requires less precision and attention by the surgeon due to the magnification and the actual incisions being performed by the robot.
In 2000, the FDA approved for one of the first robot-assisted systems to be brought to the market. The system, called the da Vinci Surgical System promised to improve the efficiency and effectiveness of medical surgeries, not just cancer-related surgeries. In order to bring the system to the market quickly, the robotic surgery system went through “premarket notification,” allowing the company to skip the rigorous safety and efficacy trials. Essentially, “premarket notification” is supposed to ensure that a device is safe and this notation helps quicken a device’s journey to the market. The FDA said that this decision was based only on short-term data and a spokesperson stated that the decision was made “based on evaluation of the device as a surgical tool and did not include evaluation of outcomes related to the treatment of cancer.” The device promises more successful surgeries with limited retraining and a smooth transition from a humancentric to robot-assisted surgery. These prospects posed limited risks and the evident benefit of improving the success rate of these surgeries; as a result, the device was approved without a thorough and holistic evaluation.
While this system has only been approved for some urological and gynecological procedures, these devices are used for a vast array of other unapproved procedures. The FDA can assess the safety of these devices for certain procedures, but they cannot prevent these systems from being used in unapproved settings in the medical field. As a result, medical professionals may still use these systems for procedures that have not been approved by the FDA.
At the beginning of March, the FDA released a statement reminding the public that robot-assisted surgeries have not been approved for mastectomy or cancer-related surgeries, two procedures for which the device is frequently used. Dr. Terri Cornelison, who works for the FDA’s Center for Devices and Radiological Health, has stated, “We are warning patients and providers that the use of robotically-assisted surgical devices for any cancer-related surgery has not been granted marketing authorization by the agency. The survival benefits to patients when compared to traditional surgery have not been established.” The FDA has claimed that there is no supporting evidence that robot-assisted surgeries are better than traditional surgeries and they have further claimed that robot-assisted surgeries result in more problems for patients receiving treatment for cervical cancer. Cornelison further states, “We want doctors and patients to be aware of the lack of evidence of safety and effectiveness for these uses so they can make better informed decisions about their cancer treatment and care.”
The FDA cited two studies that warn against the danger of robot-assisted surgery. Both studies were published by the New England Journal of Medicine. Both studies analyzed the difference between robot-assisted and traditional procedures for cervical cancer in women. The first study found that women who received surgery with robotic methods faced four times as many cancer recurrences and six times as many deaths. It should be noted that the procedure – radical hysterectomy – is considered to be a relatively safe procedure when performed correctly that can cure patients of cervical cancer. Furthermore, in the second study, 9.1% of the sample group died after minimally invasive surgeries, or in other words robot-assisted surgeries, and 5.3% died in open surgeries, which involve no robotic mechanisms.
It is not clear why robot-assisted surgeries have had worse results for cervical cancers. Dr. Pedro T. Ramirez, a surgical researcher at the Anderson Cancer Center in Houston, believes that these results may be due to the device or because carbon dioxide, which is used to provide a working and viewing space for the surgeon, may increase the spread of cancer during the procedure.
These findings by the FDA encourage patients to question their medical professionals about what type of procedures they will receive and to know the facts about different methods for surgery. In order to ensure that they receive the best care, it is important that patients have a say in the procedure they will receive by accurately weighing the risks and benefits. While the FDA cannot stop the use of these tools in the medical field, increased interest and probing of the mechanics of these systems are helping raise awareness about what is actually happening in the operation room.
Finding a Solution in the Source: Exploring the Potential for Early Beta Cell Proliferation to Disrupt Autoreactive Tendencies in a Type 1 Diabetes Model
By Reshma Kolala, Biochemistry & Molecular Biology ‘22
Residing in the pancreas are clusters of specialized cells, namely alpha, beta (), and delta cells. cells, more specifically, are insulin-secreting cells that are instrumental in the body’s glucose regulation mechanism. An elevation of the extracellular glucose concentrations allows glucose to enter cells via GLUT2 transporters, where it is subsequently metabolized. The resultant increase in ATP catalyzes the opening of voltage-gated Ca2+ channels, triggering the depolarization of the plasma membrane which in turn stimulates insulin release by cells (1). In individuals with Type 1 Diabetes (T1D), however, pancreatic islet beta cells are damaged by pro-inflammatory cytokines that are released by the body’s own immune cells. The loss of functional beta cell mass induces a dangerous dysregulation of glucose levels, resulting in hyperglycemia along with other harmful side effects. The absence of a regulatory factor in the bloodstream forces those with T1D to take insulin intravenously to remedy the consequences.
A new study led by Dr. Ercument Dirice, a Harvard Medical School (HMS) instructor and research associate at the Joslin Diabetes Center. has suggested that an increase in cell mass early in life diminishes the autoreactive behavior of immune cells towards cells, therefore halting the development of T1D (2). In a typical T1D model, the secretion of antigens from cells induces a response from the body’s immune cells. These immune cells bind to the epitopes (the recognizable portion of an antigen) that are displayed on the surface of professional antigen presenting cells (APC’s) which are littered throughout the pancreatic islets (3). This binding action induces a destructive autoimmune response to antigens secreted by cells, resulting in loss of functional beta-cell mass. It was found however that by increasing cell mass at an early age where the organs of the immune system are still developing, the immune cells stopped attacking cells.
The novel approach presented by Dirice et al. departs from the traditional method of targeting various other components involved in the destructive autoimmune response, namely APC’s or the pro-inflammatory cytokines associated with T1D progression. This method instead focuses on the source of the autoimmune marked “pathogenic” antigens, the cells themselves.
The studies were completed using two models of female non-obese diabetic (NOD) mice. One was a genetically engineered model of female mice (NOD-LIRKO) that showed increased cell growth soon after birth while the second model was done using a live mouse that was injected at an early age with an agent known to increase cell proliferation. While maintaining more than 99.5% isogenicity (4), it was found that the mice with increased cell mass had a significantly lower predisposition to develop diabetes when compared against the NOD control mice, which developed severe diabetes between 20-35 weeks of age. The study also observed the interaction between the modified cells and immune cells by monitoring the concentration of these immune cells in the spleen. In doing this, researchers were able to conclude which mice had a greater risk of developing T1D based on if mice had an abnormal increase in the concentration of these cells.
At first glance, this method appears counterintuitive as an increase in cell mass may lead one to naively assume that this would result in increased autoantigen production. This precise hypothesis illustrates the beauty of this approach. Although the specific details of this mechanism have yet to be made clear, it is believed that the rapid turnover of cells “confuses” the autoimmune reaction. The proliferated cells present unusual autoantibodies that are not observed in typical T1D progression. Dr. Rohit Kulkarni, a fellow HMS professor and researcher at Joslin noted that there is thought to be some alteration in the new cells where the autoantigens typically produced are reduced or dilated (2). As a result of the slow presentation of antigens, there is a lower proportion of autoreactive immune cells. This essentially results in a “reshapen immune profile that specifically protects cells from being targeted.” Some degree of autoimmunity would continue to exist in the body, so further immunosuppressive treatment would be required.
Early cell proliferation has been previously speculated to have a protective effect in those with reduced functional cell mass as in a Type 1 Diabetes model. Once this preventative quality is better understood, applications of this research may be further explored. Despite still being in the beginning stages, this novel approach holds tremendous potential for application to T1D if this method is able to be translated to a human model. The massive prevalence of a T1D diagnosis is illustrated by 2014 census data that states that T1D affects roughly 4.7% of the world’s adult population. Although extensive research continues to be done on several aspects of the disease, the introduction of new data by Dirice et al. may push us a small step closer to solving one of the body’s greatest metabolic mysteries.
References
- Komatsu, M., Takei, M., Ishii, H., & Sato, Y. (2013, November 27). Glucose-stimulated insulin secretion: A newer perspective. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020243/
- Yoon, J., & Jun, H. (2005). Autoimmune destruction of pancreatic beta cells. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/16280652
- Pushing early beta-cell proliferation can halt autoimmune attack in type 1 diabetes model. (2019, May 06). Retrieved from https://www.sciencedaily.com/releases/2019/05/190506124102.htm
- Burrack, A. L., Martinov, T., & Fife, B. T. (2017, December 05). T Cell-Mediated Beta Cell Destruction: Autoimmunity and Alloimmunity in the Context of Type 1 Diabetes. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723426/
- Dirice, E., Kahraman, S., Jesus, D. F., Ouaamari, A. E., Basile, G., Baker, R. L., . . . Kulkarni, R. N. (2019, May 06). Increased β-cell proliferation before immune cell invasion prevents progression of type 1 diabetes. Retrieved from https://www.nature.com/articles/s42255-019-0061-8?_ga=2.76180373.1669397493.1557550910-1092251988.1557550910
A Chemical Report on Heptachlor (Heptachlor Epoxide)
By Kaiming Tan
Author’s Note: This report analyzes and explains the biological, chemical, and environmental importance of heptachlor. More and more in today’s society, we are utilizing synthetic compounds as agricultural insecticides, which makes understanding what these chemicals do to our bodies and the environment of utmost importance. Farming strategies may seem far-removed from our daily lives, but these chemicals do not stay on the farm. They travel to our cities, to our grocery stores and markets, then make their way onto our dinner plates and into our children’s stomachs. I am constantly amazed at the power of scientific research to transform and demystify the detrimental nature of environmental and biological toxicants; this, combined with my passion in toxicology, has inspired me to research this topic and write this manuscript.
Keywords
Heptachlor, heptachlor epoxide, insecticide, environmental toxicology, biological toxin
Introduction
Heptachlor was introduced into the United States in the 1940s and 1950s, among the other chlorinated hydrocarbon insecticides, such as DDT (4). In 1971, 500,000 kilograms of heptachlor were applied to agricultural fields in soils and seeds to prevent termite infection in woods (4). However, after it became evident that heptachlor’s toxicity was a serious health concern, the Environmental Protection Agency (EPA) banned all registered use of heptachlor because of its carcinogenic properties and bioaccumulation in food and water. With the exception of treatment of fire ants in underground power transformers, there is no use of heptachlor in the United States now (2). In contrast, heptachlor is still used as an insecticide in some areas in Asia, Africa, and Eastern Europe (5). The form of greater toxicological concern is heptachlor epoxide, which is a metabolite of heptachlor in soil and the human body. Heptachlor epoxide is more toxic because it degrades slower than heptachlor, thus, is more persistent in the environment. Both heptachlor and heptachlor epoxide can be found in freshwater, estuarine, and marine systems (6). This review will cover the current literature on heptachlor toxicity and investigate the impact of heptachlor on biological systems.
Biological Fate (Absorption, Distribution, Metabolism, Elimination)
Humans and animals can be exposed to heptachlor and heptachlor epoxide through soil, air and water; however, the main route of exposure is oral, through consuming contaminated food or drinking contaminated water. Children and infants are especially vulnerable to heptachlor exposure if they consume large amounts of breast milk from their mother. For instance, if the mother’s breast milk is contaminated by heptachlor, heptachlor can be easily consumed by the infant through breast milk feeding. In human breast milk, heptachlor epoxide was detected ranging from 0.13 to 128 ppb (parts per billion) (8). Another route of exposure is through inhalation. When heptachlor is deposited into the soil, it becomes heptachlor epoxide, which then spreads into the air. As a result, workers who use heptachlor to kill termites are potentially susceptible to inhaling heptachlor epoxide from the air (2). In addition, touching contaminated soils causes exposure to heptachlor dermally (1).
Heptachlor persists in the environment after agricultural application. Even though there was no information on the background levels of heptachlor and heptachlor epoxide in the air or soil, there are 20 to 800 ppt (parts per trillion) heptachlor in drinking water and groundwater in the United States (2). Contaminated fish and shellfish were detected with levels of 2 to 750 ppb of heptachlor and 0.1 to 480 ppb of heptachlor epoxide. To date, there is no available data regarding the biological fate of heptachlor on humans. However, in rats, at least 50% of the orally-administered dose of heptachlor is absorbed in the gastrointestinal tract.The absorption is indicated by the presence of heptachlor and/or heptachlor epoxide in the rats’ liver (9). When heptachlor epoxide enters the bloodstream after absorption, it becomes the predominant metabolite in the body of laboratory female rats. Heptachlor epoxide in the bloodstream is positively correlated to the heptachlor dose administered (10). In other words, the higher heptachlor dose one consumed, the more heptachlor epoxide would appear in the bloodstream.
Because of heptachlor epoxide’s high lipophilicity (likelihood in dissolving fats), its residue is found highest in adipose tissue in human infants (0.32±0.10ppm), while undetectable in the brain. These data also suggest that heptachlor epoxide can be transferred between pregnant women and their babies through the placenta (11). Furthermore, heptachlor and heptachlor epoxide can be stored in human breast milk. Jonsson et al.’s study in 1977 sampled the breast milk of 51 women who had no previously known exposure to heptachlor and detected an average concentration of heptachlor epoxide between 0.0027ppm and 0.019ppm (12). To the researchers, the results proved worrisome as the women’s breast milk contained high levels of heptachlor, which could damage the infant. The results were even more of a concern because there was no known interaction between the women studied and heptachlor.
After heptachlor is absorbed in the body, the primary metabolite of heptachlor in humans and rats is heptachlor epoxide. Heptachlor and heptachlor epoxide are usually metabolized by Cytochrome P450 (CYP450) enzymes. CYP450 enzymes are the body’s major class of enzymes responsible for detoxification. Moreover, heptachlor alters liver function by increasing gluconeogenic enzymes’ activities, which upregulates glucose synthesis from glycogen. Since most of heptachlor is metabolized in the liver, humans with liver diseases may have increased bioaccumulation of heptachlor epoxide because of decreased CYP450 enzyme activity (13). Despite the metabolism of heptachlor in the liver, most heptachlor epoxide can be stored in the adipose tissue because of its high lipophilicity and long half-life (1).
In terms of excretion, orally-administered heptachlor in rats was excreted in the form of heptachlor epoxide and also as heptachlor. Radiolabeled heptachlor epoxide is excreted ten times more in the feces compared to urine after ten days of oral administration in rats, due to the high lipophilicity of heptachlor metabolites (8).
Mechanism of Action
The primary organ and organ systems that heptachlor targets are the liver, central nervous system and reproductive system, while secondary target organs include kidneys and lungs. Heptachlor is primarily metabolized in the liver into heptachlor epoxide, which has the same toxic potential. The reproductive system is a sensitive target for heptachlor toxicity because this system lacks a comprehensive detoxification system like the CYP450 enzymes in liver. CYP450 enzymes facilitate chemical reactions that chemically convert the toxicant into a more hydrophilic metabolite, thereby enhancing toxicant elimination by urine. Oral exposure of 1.8 mg/kg/day to female rats over 14 days caused decreased fertility due to decreased estradiol and progesterone levels. Estradiol and progesterone are important hormones produced by the ovaries, placenta, and adrenal glands; decreased levels may result in endocrine disruption and delayed sexual development. Oral exposure of 0.65 mg/kg/day over 70 days caused decreased sperm count in male rats. The mechanism of heptachlor’s reproductive toxicity remains unknown (1-2).
GABA, the molecule that normally binds the GABA receptor, is an amino acid which can also serve as an inhibitory neurotransmitter in the mammalian brain. Heptachlor primarily acts as a noncompetitive antagonist of the chloride channels of the GABAA receptors in mammals. In other words, heptachlor blocks the Chlorine channel in the cells thus preventing GABAA activation (1-2, 14). By inhibiting the inhibitory neuron, heptachlor causes hyperexcitability of the cells. Although the heptachlor toxicity mechanism in liver is unknown, it is observed that in rats that presence of heptachlor elevates intracellular calcium levels and induces protein kinase C (PKC), which in turn activates the activator protein-1 DNA binding thereby causing tumorigenesis (2, 14).
When animals are exposed to heptachlor above the toxic threshold, symptoms occur including tremors, convulsions, ataxia, and changes in EEG patterns (1). Also, rats who were orally exposed to >320 ppm of heptachlor had consistently lower body weight than rats that were unexposed (15). In humans, clinical symptoms of heptachlor toxicity include seizure, vomiting, and convulsions (2).
Although heptachlor epoxide is theoretically an effective mutagen because of its high reactivity as an electrophile, research studies on heptachlor mutagenicity yielded mostly negative results. For example, heptachlor was not mutagenic in the auxotrophic strains of E. coli by reversion bioassays (14,15). In addition, heptachlor is proven to increase liver tumor incidence as an epigenetic promoter in previously initiated B6C3F1 mice. Heptachlor is a carcinogen in mice by inhibiting intercellular communications to promote tumor growth (14,16). On the other hand, research studies that investigate heptachlor’s teratogenicity, or tendency to disturb embryonic development, yielded mostly negative results. For instance, although feeding rats with diets containing heptachlor produces pups with higher mortality, no congenital malformation was found (2, 14, 17).
In male rats, oral LD50 (lethal dose for 50% of the study population) of heptachlor is 40 to 100 mg/kg body weight based on data from two studies. In contrast, the oral LD50 of heptachlor in male chickens is 62 mg/kg body weight (1, 17). Symptoms displayed by heptachlor acute toxicity in animal subjects include hyperexcitability, tremors, convulsions and paralysis (18).
Long-term exposure to heptachlor can cause liver damage in animals. In rats, prolonged exposure of sub-lethal doses of heptachlor is associated with increased CYP450 enzyme induction and other hepatic microsomal enzymes, in addition to liver hypertrophy (18). In humans, chronic exposure to heptachlor results in storage in adipose tissue and breast milk, because heptachlor is very lipophilic. Infants are at risk of being exposed to large doses of heptachlor if they consume contaminated breast milk (17). Despite the lack of human studies on long-term exposure, a multi-generation study conducted on rats concluded that oral exposure of 6 mg/kg/day of heptachlor is associated with decreased litter size, increased mortality and lens cataract (2). Because of its high lipophilicity, heptachlor residues can remain in the body over time. Heptachlor’s long-term toxicity damages the body gradually, further underlying the need for strict regulations of heptachlor use worldwide.
Overview of Latest Research
Recent research on heptachlor focuses on its toxicity, specifically on the oxidative stress caused by heptachlor and its metabolism in aquatic animals. For example, Vineela et al.’s study investigates the impact of sub-lethal concentrations of heptachlor on carp fish Catla catla by measuring enzymatic activities of mostly Phase II enzymes (19). Phase II enzymes, a part of the CYP450 enzyme class, primarily perform conjugation reactions to convert the chemical into more water-soluble form to enhance urine elimination. Oral exposure of heptachlor at 1.46mg/L (20% of LC50 concentration) for 45 days causes a significant increase in lipid peroxidation, superoxide dismutase, glutathione-S-transferase and catalase activity in Catla catla. The results suggest that carp fish have a sensitive biological defense system against heptachlor, because low concentration activates the detoxification by increased biomarkers of primarily Phase II activity to prevent heptachlor toxicity and organ damage (19).
In addition to heptachlor toxicity, current research also focuses on how to remove existing heptachlor from the environment via microbial degradation, as this method is more environmentally mindful and cost-effective than current physicochemical methods. Qiu et al.’s study discovered a novel strain of bacteria, named strain H, that can metabolize heptachlor efficiently. Strain H is a Gram-negative, short rod-shaped, single-cell bacterial strain that can degrade heptachlor at a rate of 88.2% degradation in 130 hours when exposed to 300 μg/L of heptachlor at 30oC. The main metabolites of heptachlor by strain H include heptachlor epoxide, chlordane epoxide, and 1-hydroxychorodene. This innovation allows possible bioremediation by microorganisms like strain H in heptachlor-contaminated soil and water to reduce heptachlor toxicity and threat to the environment and animals (20).
Recent studies on heptachlor took a creative approach in gaining a deeper understanding in reducing heptachlor toxicity by enzymatic activity in aquatic model organisms and inventing novel microorganisms to metabolize heptachlor into less toxic metabolites. These new techniques will benefit public health by developing cost-effective ways for toxicant removal and controlling the environmental/biological fate of toxicants without causing additional harm.
Conclusions
When initially used as an insecticide, heptachlor’s toxicity became a threatening health concern. The ban of commercial heptachlor use in the United States was a step in the right direction, as misuse of heptachlor can cause severe environmental consequences such as prolonged residue in soil and water habitats along with toxicities in humans and animals. Based on this research, other countries should pursue a substitute pesticide that has less potential for environmental and biological damage than heptachlor.
Most studies conducted regarding heptachlor toxicity were conducted between the 1950s and the late 1980s. Future research can focus on the dose-response relationship of heptachlor exposure within large populations and whether genetic polymorphism contributes to the metabolism of heptachlor. Environmental toxicologists can also study the synergistic toxicity of heptachlor on environmental damage with other pesticides, since common pesticides often contain more than one chemical (1,2).
Acknowledgment
The author would like to thank Dr. Matthew Wood and Mr. Thomas Sears for providing feedback on early versions of this manuscript.
Works Cited (in order of appearance)
- Reed, N.R., & Koshlukova, S. (2014). Heptachlor. In Encyclopedia of Toxicology (pp. 840-844).
- United States. Agency for Toxic Substances Disease Registry, & Syracuse Research Corporation. (2007). Toxicological Profile for Heptachlor and Heptachlor Epoxide.
- Ivie, G., Knox, W., Khalifa, J., Yamamoto, R., & Casida, S. (1972). Novel photoproducts of heptachlor epoxide, Trans -chlordane, and Trans -nonachlor. Bulletin of Environmental Contamination and Toxicology, 7(6), 376-382.
- Hodgson, E. (2004). A textbook of modern toxicology (3rd ed.). Hoboken, N.J.: Wiley-Interscience.
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The Effect of Trastuzumab on HER2-Signaling in Breast Cancers to Induce Cardiotoxicity
By Karissa Cruz, B.S. Biochemistry and Molecular Biology, Spring ‘19
Author’s Note: I wrote this piece as part of my UWP 104F assignments and ended up becoming really interested in what I wrote about. I specifically chose this topic because I think breast cancer is a smart, complex disease, and the treatment can change day-to-day. I wanted to shed light on a widely accepted breast cancer treatment that is now under review after discovering that it can cause cardiac dysfunction.
Lethal and Perplexing: An Overview of Metal Phosphides and Their International Impact
By Taylor Riedley, Biology, ‘20
Author’s Note: I wrote this paper for my UWP 102B class with Dr. Brenda Rinard in Winter Quarter 2019. For the preliminary stage of an assignment, I reviewed primary research on metal phosphide poisoning and wrote a formal paper geared towards a medical audience. For the final stage, I translated my formal paper into this one, intending it for a curious though not necessarily scientific audience. I chose this topic because I am considering an occupation in forensic pathology, and while I searched for articles about pathological findings, I came across an autopsy of a victim of metal phosphide poisoning. I was struck by the discrepancy between the general outward symptoms and the lethal organ damage of phosphide patients. I was further appalled when I learned that metal phosphides are within ready reach of children. I viewed this paper as an opportunity to draw attention to the need for more research on metal phosphides, and especially in this more accessible incarnation, to encourage readers to advocate for those who are not given the information necessary to stay safe.
Why Your Dog is Itchy: A Review of Common Skin Allergies
By Jeffrey Nguyen, Animal Biology, ‘19
Author’s Note: I originally wrote this piece for UWP 104E: Writing in the Sciences. The assignment called for an explanation of any scientific topic to the general public and I thought to write on a topic that would be both useful and relatable to pet owners. Skin allergies affect dogs of all breeds and can bear severe consequences if left untreated. I hope that this paper increases awareness on animal health and convinces pet owners to consider taking a second glance in those moments they observe something out of the ordinary.
Davis Downfall According to Sierra Club
By Mari Hoffman
Authors note: UC Davis does an immense amount of waste reduction and energy conservation practices, but after writing this essay in my Water Quality at Risk class I wondered if it is enough. In 2018, UC Davis did not make it on the “Cool Schools” list by Sierra Club Magazine. The previous year we were ranked as number 32. I was assigned this essay to compare some of the practices that UC Davis does with other schools to get an understanding on what we are excelling at and where we can improve. I chose to focus on how we can improve in regards to water waste with an implementation of porous concrete.
A Regenerative Cocktail: Combination of Drugs Promotes the Conversion of Glial Cells to Neurons
By Reshma Kolala, Biochemistry & Molecular Biology ‘22
Author’s Note: While browsing recent findings in Neuroscience, I came across research investigating the possible conversion of glia to neurons. Although the conventional idea that neurons are irreplaceable has been overturned in multiple research studies, I was immediately intrigued by the possibility for neighboring glia to be the source of neural regeneration. The implications of this research could completely transform how treatment is approached in the neuroscience field of medicine.