PS1 – Data Visualization

This article is Chapter I from the author’s book Statistics and Probability Flashcards.


Individuals and variables

In a dataset, the individuals are the items with one or more properties, called variables. Individuals can be events, cases, objects, people, etc.

Student (individuals) Height (variables)
John 190 cm
Ali 175 cm
Paul 165 cm
Clara 160 cm

Table 1.1. example of a data set with items and variables.

Individuals and variables are called data. Table 1.1 is called a data table.

Here’s another example of a data table containing other variables:

Student Height Weight Likes football
John 190 cm 100 kg Yes
Ali 175 cm 90 kg No
Paul 165 cm 60 kg No
Clara 160 cm 63 kg Yes

Table 1.2. example of a data set with items and more than 1 variable category.

Variables can be categorical or quantitative. In table 1.1 there’s one quantitative variable: the height whereas in table 1.2 there are two quantitative variables (height and weight), and one categorical variable (likes football).

Quantitative variables are numerical variables: counts, percents, or numbers.

Categorical variables are non-numerical variables. Their values aren’t represented with numbers: words, not numbers.

This data set presented in table 1.1 and table 1.2 is called one-way data because we have just a single individual (item) that has one or many properties attached to it.

How to build a data table?

When you build a data table, it is important to think about whether you have more individuals or more variables.

In tables 1.1 and 1.2 the number of individuals listed was greater than the number of variables. If we have many variables but only a few individuals, it is advisable to list the individuals across the top and the variables down the left side.

John Ali
Height 190 cm 175 cm
Weight 90 kg 75 kg
Likes football Yes No
Likes pizza Yes Yes

Table 1.3. Since the number of variables is bigger than individuals, listing the variables vertically would make the data table more appropriate than if we had tried to list all the variables horizontally.

Data visualization

Bar graphs and pie charts

Two of the simplest ways to summarize and graphically represent data are bar graphs and pie charts.

Bar graphs apply a series of rectangular bars to show absolute values or proportions for each of the data categories whereas pie charts show how substantial each data category represents as a part or proportion of the whole, by using a circular format with different-sized “slices” for different percentages of the total.

Rank Country Oil production (bbl/day)
01 USA 15,043,000
02 Saudi Arabia (OPEC) 12,000,000
03 Russia 10,800,000
04 Iraq (OPEC) 4,451,516
05 Iran (OPEC) 3,990,956
06 China 3,980,650
07 Canada 3,662,694
08 United Arab Emirates (OPEC) 3,106,077
09 Kuwait (OPEC) 2,923,825
10 Brazil 2,515,459

Table 1.4. Top 10 world Oil producers (“Production of Crude Oil including Lease Condensate 2019” U.S. Energy Information Administration)

Figure 1. Bar chart – Top 10 world Oil producers (“Production of Crude Oil including Lease Condensate 2019” U.S. Energy Information Administration)

Notice that we have a list of the Oil producers (countries) across the bottom of the bar graph, with the count of the Oil production (bbl/day) up the left side.

The countries are the individuals, and the count is a quantitative variable because it represents the numeric property of each of the individuals. The bar graph is one of the best ways to represent this data because it is possible to get quickly an overview of which countries produce the most oil.

Figure 2. Pie chart – Top 10 world Oil producers (“Production of Crude Oil including Lease Condensate 2019” U.S. Energy Information Administration)

Now we can quickly see that the United States produces the most of the total oil daily, biggest than any other country, Saudi Arabia occupies second place, and Brazil is the 10th world’s biggest oil producer.

Venn diagrams

A Venn diagram is a diagram that shows all possible logical relations between a finite collection of different sets from a two-way table.

Good Cheap Fast Total
Expensive 10 0 10 20
Low quality 0 10 10 20
Slow delivery 10 10 0 20
Best choice 10 10 10 30
Other 20 20 20 60
Total 50 50 50 150

Table 1.5. two-way data table

Figure 3. Venn diagram

Box-and-whisker plots

Box-and-whisker plots (also called box plots) are a great method for graphically depicting groups of numerical data through their quartiles. It is very useful when you want to show the median and spread of the data (see chapter IV) at the same time.

Assuming that we have the following data set: [1, 2,2, 2, 3, 3, 4, 6, 8,8, 10, 11, 11, 16]:

Figure 4. Box-and-whisker chart

The horizontal line in the center of the box is the median of the data set, so the median of the data set represented in the chart above is 5.

The dot at the end of the bottom whisker is the minimum of the data set, and the dot at the top of the right whisker is the maximum of the data set. So in this plot, we can say that the minimum is 1, that the maximum is 16, so the range would be 16 − 1 = 15.

The IQR (interquartile range) is given by the ends of the box. Since the box above extends from 2 to 10.25, the IQR is 10.25 − 2 = 8.25.

We can summarize the information given by the Box-and-whisker chart above in the following table:

Min Q1 Median Q3 Max
1 2 5 10.25 16

Dietary supplements and functional foods

Food supplements are foodstuffs intended to supplement the normal diet, the constitute a concentrated source of nutrients or other substances having, alone or in combination, a nutritional or physiological effect. They are marketed in many forms (capsules, pastilles, tablets, powder packets or ampoules).

The nutritional or health claims they claim have, since July 2007, been very strictly regulated under the European Regulation 1924/2006, which requires scientific proof to be provided to the European Medicines Agency (EMA).

Functional foods are not defined by legislation. They are considered common foods intended for consumption as part of a balanced and varied diet. Their particularity lies in the fact that they contain biologically active compounds that have beneficial effects on one or more target functions of the body, beyond the basic nutritional effects, in order to improve health and well-being and/or reduce the risk of disease.

Dairy products, especially yogurts, are the most abundant probiotic foods, with Danone’s Activia® and Actimel® products leading the way.

Like many foods, functional foods and probiotics are subject to safety and labeling rules, in particular with regard to claims used by the food industry as a selling point.

Recently, new guidelines have tightened the regulations around these probiotic foods because their health benefits were difficult to recognize.

European Union Regulation No 432-2012 of 16 May 2012 establishes a list of authorized health claims on foods and specifies that health claims must be based on generally accepted scientific evidence.

Probiotics fall into two types of claims: function claims and therapeutic claims:

Claim Any representation that states, suggests or implies that a food has particulate qualities related to its origin, nutritional properties, nature, processing, composition or any other quality.

Health claim refers to any representation in labeling and advertising that states, suggests or implies that there is a relationship between the consumption of a food or food constituent and a person’s health.

Functional claim refers to a health claim that describes the physiological effects of food or food constituents on the body’s normal functions or biological activities associated with health or performance. Functional claims can be made about the physiological effects of probiotic microorganisms in foods (e.g., “promotes regularity” and “improves nutrient absorption and aids digestion”). Function claims must include a specific, scientifically substantiated physiological effect associated with good health or performance and providing useful information to consumers.

Therapeutic claim refers to the treatment or mitigation of a disease or health disorder or related to the recovery, correction or modification of bodily functions. For example,”[name of food or food constituent] lowers blood cholesterol”.

The assessment of probiotics for food use is described in the report of the Joint FAO/WHO Expert Consultation (Food and Agriculture Organization of the United Nations and World Health Organization).

Specific labeling guidelines are outlined in the Canadian Food Inspection Agency’s (CFIA) Guide to Food Labelling and Advertising, which applies to all products containing probiotic microorganisms. According to the appropriate description of a probiotic product, as indicated on the label, should include the following points:

  • Strain Identification: Any claim for a probiotic must be accompanied by the Latin name of the microorganism (i.e. genus and species), as well as the name of the strain of the microorganism. For consistency, it is recommended that the strain should be identified by the number assigned by an internationally recognized culture bank, such as the American Type Culture Collection.
  • Quantity declaration: The quantity of the probiotic microorganism(s) present in the product must be indicated in colony-forming units (CFU) in a specified portion of the food. This statement must appear next to the Nutrition Facts table or ingredient list, or in close proximity to the claim.
  • List of ingredients: Any food containing probiotic microorganism(s) must display a list of ingredients in accordance with the sections of the Food Regulations. The probiotic microorganism must be designated by its common name or by the class name.


The notion of probiotics has recently developed and most pharmacists have not been trained in these new food supplements.

From birth, our gastrointestinal tract is colonized by many microorganisms that constitute the digestive microbiota. This complex and diversified ecosystem, unique to each individual, contributes to the proper functioning of the intestine through the many activities it carries out. However, the balance of the microbiota is sensitive and its rupture occurs in the pathophysiology of various intestinal disorders, hence the idea of positively modulating a microbiota unbalanced by the administration of probiotics.

The term “probiotic” means “for life” and refers to living microorganisms that, when ingested in appropriate amounts, produce a benefit to the health of the host that goes beyond basic nutritional functions.

Probiotics are often lactic acid bacteria (lactobacilli and bifidobacteria) or yeasts introduced into the diet in the form of fermented milk products or food supplements.

These microorganisms strengthen the intestinal and vaginal flora. Their presence makes it possible to fight against the proliferation of pathogenic bacteria.

Several clinical studies have already demonstrated the efficacy of certain probiotics in the treatment of systemic and infectious diseases such as acute diarrhea and Crohn’s disease.

Other studies have suggested a potential application for the treatment of urogenital infections, colon cancer, atopic dermatitis and allergic diseases including food allergy such as lactose intolerance.


The definition of probiotics has evolved over time according to researchers, scientific knowledge and technological advances.

In the 20th century the Nobel Prize winner, Elie Metchnikoff, observed that a surprising number of people in Bulgaria lived for more than 100 years. This longevity could not be explained by the advances in modern medicine, because Bulgaria, one of the poorest countries in Europe at that time, did not benefit from such advances. Dr. Metchnikoff found that Bulgarians consume large quantities of yogurt, he associated the increase in longevity observed with the consumption of living microorganisms from fermented dairy products. Although Metchnikoff saw germs as rather harmful to human health, he considered it beneficial to replace bacteria in the gastrointestinal tract with yogurt, including the Bulgarian bacillus. He then explained the better beneficial effect of this bacteria by the absence of alcohol production (harmful to longevity), compared to bacteria present in other fermented milk such as kefir or kumys. In addition, he assumed that the lactic acid produced, as well as other unidentified factors, would act synergistically to inhibit the growth of putrefaction bacteria in the colon.

At the same time, in 1906, the French pediatrician Henry Tissier observed that the stools of children with diarrhea contained a small number of bifidobacteria compared to the stools of healthy children. He then suggested that these bacteria be administered to diarrheal patients to help them restore a healthy intestinal microbiota.

Metchnikoff and Tissier are therefore the first to put forward the idea of administering exogenous microorganisms to compensate for a possible dysfunction in our intestinal ecosystem. The concept of “probiotics” was born.

Nevertheless, it was not until 1954 that the term probiotics was introduced into the literature by Ferdinand Vergin in a paper entitled “Anti-und Probiotika”. This term derived from the Greek “pro bios”, which literally means “for life” as opposed to the harmful effects of antibiotics

In 1965, Lilly and Stilwell, in the journal Science, defined probiotics as substances produced by microorganisms capable of stimulating the growth of other microorganisms.

In 1989, Fuller highlighted the microbial nature of probiotics by redefining the term as a “living microbial nutritional supplement that has a positive effect on the host animal by improving its intestinal balance”.

In 1992, Havenaar and Huis in’t Velt further refined the term to “a viable culture composed of one or a mixture of bacteria that, when applied to animals or humans, has a beneficial effect on the host by improving the properties of native flora. ».

In 1998, Guarner and Schaafsmaa specified that probiotics are “living microorganisms, which, when consumed in adequate amounts, have a beneficial effect on the health of the host”.

In 2002, the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) formalized the definition of probiotics to avoid any drift.

Probiotics are therefore defined as “living organisms that, when ingested in sufficient quantities, have a beneficial effect on the health of the host”.

History, therefore, underlines that the current definition could still evolve, as there are still many fields of research to better understand and understand the action of probiotics.


The conditions and marketing authorization of probiotics are defined according to their drug or food application. Most probiotics are functional foods or are used as food supplements. These “healthy foods” are at the border between the drug and the traditional food and are governed by food legislation.

Probiotic foods

The global market of probiotic foods has been growing rapidly since the early 2000s, particularly in Europe. This dynamic is supported in particular by the link between food and health benefits.

Probiotics used as food supplements, as well as functional foods, are considered as food and are governed by the relevant legislation. They are different from dietary foods that are intended for a particular food and require a specific formulation or manufacturing process to differentiate them from the common food, and from medicinal products.

Probiotics for Chronic Constipation

There are approximately 3.8·1013 single-celled microorganisms for every single “reference man”  weighing 140 pounds, and their total mass is about 200 grams (

Intestinal microbiota aids in the breakdown of food products into absorbable nutrients, stimulates the host immune system, suppress inflammation [source], prevents the expansion of pathogenic bacteria and produces a substantial variety of biologically major compounds such as short-chain fatty acids that nourish the gut.

Gut Bacteria
Gut Bacteria

The concept behind probiotics was introduced in the early 19th century when Nobel laureate Elie Metchnikoff (1845–1916), known as the father of probiotics <fn>Front Public Health. 2013; 1: 52. Published online 2013 Nov 13. Prepublished online 2013 May 30. doi: 10.3389/fpubh.2013.00052 </fn>.

Elie Metchnikoff (1845–1916)
Elie Metchnikoff (1845–1916)

Probiotics are given or attenuated microorganisms defined as, when administered in adequate amounts, being able to confer health benefits on their host when they are given in.

Lactobacillus acidophilus, for example, lives in our digestive, urinary and genital systems and can be found in some fermented foods like yogurt. It may help reduce cholesterol, prevent Diarrhea, prevent Vaginal Infections, promote Weight Loss and improve symptoms of irritable bowel.

Lactobacillus acidophilus
Lactobacillus acidophilus

Studies suggest that taking probiotic supplements may shift the balance of gut bacteria in a way that increases your body’s defenses against allergies, infections, and cancer.

Probiotics and constipation

Chronic constipation is a symptom-based gastrointestinal disorder most common among the elderly, it is characterized by bowel movements that occur more frequently than normal. Constipation is most likely multifactorial. According to Harvard Medical School, it is more frequent than diarrhea and affects approximatively 14% of adults each year.

Researchers have shown an increased interest in the potential therapeutic applications of Probiotics to prevent or treat a variety of health problems including constipation and diarrhea. They also found a favorable effect in stool consistency and relief in abdominal discomfort making them increasingly used as alternative treatment options.

Although Probiotics have still been widely used nowadays for the treatment of constipation, the industry still has serious concerns about the long-term safety of probiotics which remain still partly unclear.

Mechanism of action

Several mechanisms have been proposed by which probiotics may benefit chronic constipation. Probiotics may modify the altered intestinal microbiota, and may eventually influence gut sensory-motor functions (Kawabata et al.). A healthy gut can benefit the regularity of your digestion as well as cognitive function like mood. One in vitro study (Bar et al.) suggested that Escherichia coli Nissle 1917 Supernatants (clear liquid overlying material deposited by settling, precipitation, or centrifugation) (Mutaflor®) could effectively enhance colonic contractility by direct stimulation of smooth muscle cells.

A recent study reported that methane and carbon dioxide, which are principal end products of bacterial fermentation could increase stool bulk and promote colonic transit (Lopez).

Which probiotic strains are best for constipation?

Proven probiotics available for constipation and bloating in adults are:

  • Lactobacillus casei Shirota (or L. Casei), which has effects on constipation and stool hardness, but not necessarily on flatulence and bloating
  • Lactobacillus plantarum LP01 (or L. Plantarum), which would facilitate stool evacuation
  • Bifidobacterium breve BR03 (or B. Patent), which would also improve the consistency of stool, helping to expel it
  • Bifidobacterium lactis DN-173 010 (or B. Lactis)
  • Bifidobacterium lactis B94 
  • Bifidobacterium lactis HN019
  • Bifidobacterium longum W11 (or B. Longum) 
  • Escherichia coli Nissle 1917 (or E. Coli), which allowed treated patients to go from 2 to 6 trips to the toilet per week

Proven probiotics available for constipation and bloating in children are:

  • Lactobacillus reuteri (or L. Reuteri), which has been shown to have an effect on chronic constipation in infants
  • Lactobacillus Casei rhamnosus Lcr35 or L, Casei), which relieves constipation while reducing abdominal pain

Health Benefits of Goat Milk

Like cow milk, goat milk is technically considered a type of dairy because it’s produced from a mammal. Its high in many necessary nutrients and is a good source of vitamins and minerals like calcium Ca, phosphorus P. Goat milk also contains medium-chain fatty acids MCFAs that have 6–12 carbon atoms which are considered as ‘natural fuel’ for the body and brain.

Health Benefits of Goat Milk
Health Benefits of Goat Milk

Nutritional information

Amount Per 100 grams

Energy: 69 Calories
% Daily Value*
Total Fat 4,1 g 6%
Saturated fat 2,7 g 13%
Polyunsaturated fat 0,1 g
Monounsaturated fat 1,1 g
Cholesterol 11 mg 3%
Sodium 50 mg 2%
Potassium 204 mg 5%
Total Carbohydrate 4,5 g 1%
Dietary fiber 0 g 0%
Sugar 4,5 g
Protein 3,6 g 7%
Vitamin A 3% Vitamin C 2%
Calcium 13% Iron 0%
Vitamin D 12% Vitamin B-6 0%
Cobalamin 1% Magnesium 3%
* Percent Daily Values are based on a 2,000 calorie diet. Your daily values may be higher or lower depending on your calorie needs.

Health benefits of goat milk

In spite of the fact that cow’s milk dominates in the US, goats milk is actually the world’s preferred milk. <fn>Live Strong</fn>

  1. Goat Milk is Easier to Digest
  2. Contains Fewer Allergens and Less Inflammatory
  3. It is High in Calcium
  4. It Helps Reduce Cholesterol Levels
  5. It Promotes Glowing Skin
  6. It Enhances Nutrient Absorption

Easier to Digest

Goat milk is better for digestion because it contains smaller Fat Molecules. Thus, it doesn’t require homogenization, the small fat molecules do not separate and remain suspended in the cream, as a result, they cause less stress on our digestive processes.

Goat and sheep milks have almost identical protein structure, but when compared to the protein in cow milk, they both contain remarkably less of the alpha S1 casein protein, which is connected to allergies.

Recent studies have revealed that goat milk may be a hypoallergenic alternative to cow milk, particularly for children. <fn></fn>

Goat milk contains slightly less lactose than cow milk (4.1 % compared to 4.7 %) which may give it a mild advantage in terms of digestion. The fermentation process used to produce cheese and yogurt also reduces the lactose, which explains why some individuals with lactose issues can enjoy certain fermented dairy products. The longer a cheese is aged, the less lactose it will contain.<fn></fn>

Fewer Allergens and Less Inflammatory

Cow milk allergy symptoms are often caused by one of the proteins found in it like A1 casein which is highly inflammatory for some people and may cause allergic reactions, especially in children. Allergy symptoms can range from hives and runny noses to abdominal cramps and colic. Goat milk is more similar to human milk, it contains 20% fewer allergens than cow milk making it an ideal alternative for people who experience allergic reactions to cows milk, as they might not be affected.

High in Calcium

While cow milk is frequently touted as one of the main foods high in calcium there’s no need to worry about not getting sufficient necessary quantity of calcium when switching to goat milk. Goat and cow milk might point on the scale similarly for mineral content. Goat’s milk contains about 33 % of the daily recommended value in one cup versus 28 % in cow milk.

(Image: carafe/amanaimagesRF/amana images/Getty Images)
(Image: carafe/amanaimagesRF/amana images/Getty Images)

Goat Milk Reduce Cholesterol Levels

Goat milk assists increase good cholesterol levels in the blood.


Goat milk is fast becoming one of the most sought after body-care products on the planet and with good reason, goat milk is straightforwardly absorbed by the skin bringing with it moisture restorative proteins, vitamins, and minerals that help keep your skin soft.

The lactic acid found in goat milk helps rid your body of dead skin cells and promotes skin smoothness and thickness, parallelly, it increases metabolism and prevents toxins from accumulating in the skin cells.

Enhancing Nutrient Absorption

Aside from the bounty of minerals nutrients and vitamins that goat milk offers its nutritional benefits don’t end there. Like mentioned above, one of the primary advantages of goat milk is that it’s chemical composition is far closer to human milk than cow milk is, which means our bodies can absorb and process more of the milk’s nutrients compared to cow milk and it taxes our digestive system far less.

Best Essential Oils and Their Health Benefits

An Essential oil is a concentrated hydrophobic liquid containing volatile – at normal temperatures – chemical compounds from plants. The term ‘essential‘ does not mean ‘necessary‘ or as with the terms essential amino acid or essential fatty acid which are so-called since they are nutritionally required by a given living in contrast to fatty oils.

Before the discovery of distillation, all essential oils were extracted directly from biomass (the bark, flowers, fruits etc.). They have been used therapeutically for thousands of years by Greeks Romans and Egyptians to remedy everything from skin conditions and injuries to fever and so many other diseases.


Egyptians and Phoenicians Jews and Arabs, Indians and Chinese greeks and Romans and even Mayas and Aztecs all possessed innovative extraction processes, maceration, alembic distillation, etc. Hydrodistillation of fresh plant material is the most used technique to isolate essential oils.


Hydrodistillation is an ancient technique for the extraction of essential oils, it is still being applied in several sectors such as food cosmetics and pharmaceutical industry.

Raw plant material such as flowers leaves wood bark roots seeds and peels are extracted by water distillation whilst soaked and boiled with water in a distillation apparatus for hydrodistillation. The mixture is heated and volatile materials are carried away. Most oils are distilled in a single process. One exception is ylang-ylang (Cananga odorata) which requires a purification step through fractional distillation.

The recondensed water (plant water essence) may be sold as another fragrant product.

Other techniques

Other methods have been developed and introduced to extract Essential Oils :

Mechanical/Cold expression

Solvent extraction

Ethanol is the most common bio-solvent obtained by the fermentation of sugar-rich materials such as sugar beet and. Its outstanding feature is the easy manipulation of the dielectric constant value of water that can be made to vary over a wide range just by changing the temperature and pressure. Ethanol is used to extract fragrant compounds from dry plant materials as well as from impure oils or concretes that have been produced firstly by organic solvent extraction expression or enfleurage. For instance, the extraction yield of essential oils from Japanese citrus was increased by 44% compared to the traditional extraction methods <fn></fn>. Problems of the traditional extraction techniques such as steam distillation lie in the huge quantities of plant material which are required to extract essential oils on a commercial scale.

Florasols extraction

Essential Oils production

According to various economic analyses, growth will continue and by the 2020s production is expected to reach 370,000 tonnes annually and be valued at more than $10 Billion USD (current dollars) <fn></fn>

World production of essential oils (000 t; 000,000 USD). Source: EFEO, ISMEA.
World production of essential oils (000 t; 000,000 USD). Source: EFEO, ISMEA.

Best essential oils and their health benefits

Whichever oils you choose to use ensure that when purchasing said oils, you get the ones that are organically produced and that they don’t include chemicals obtained during processing. Here’s a list of 10 popular essential oils whose benefits are mostly centered on disease prevention. <fn>Web site:</fn>

  • Peppermint: Used to boost energy and help with digestion.
  • Lavender: Used for stress relief.
  • Sandalwood: Used to calm nerves and help with focus.
  • Bergamot: Used to reduce stress and improve skin conditions like eczema.
  • Rose: Used to improve mood and reduce anxiety.
  • Chamomile: Used for improving mood and relaxation.
  • Ylang-Ylang: Used to treat headaches, nausea and skin conditions.
  • Tea Tree: Used to fight infections and boost immunity.
  • Jasmine: Used to help with depression, childbirth, and libido.
  • Lemon: Used to aid digestion, mood, headaches and more.

Fear of Poison

Fear of Poison or Toxicophobia may be defined as an overwhelming and debilitating fear (phobia) of being poisoned by contact or ingestion of ‘imaginary’ poisons or toxic products, in food for example (Garnier-Del. 1972). <fn>The genetic basis of panic and phobic anxiety disorders.Smoller JW1, Gardner-Schuster E, Covino J. Am J Med Genet C Semin Med Genet. 2008 May 15;148C(2):118-26. doi: 10.1002/ajmg.c.30174.</fn>


The origin of this specific phobia is not known, but like other types of phobia, a combination of environmental factors (trauma related to the object of the phobia) and hereditary factors should explain the occurrence of the phenomenon. Genetic epidemiologic studies have documented that these disorders are familial and moderately heritable<fn></fn>. Many specific phobias are primarily initiated by a stressful triggering event, such as a traumatic event in childhood, or the association of a personal tragedy with the object of the phobia.

fear of poison

Symptoms of Toxicophobia

The symptoms of toxicophobia are similar to the symptoms generally present for any phobia:

  • Anxiety, even anguish at the sight of the phobic object
  • Tachycardia caused by panic and fear
  • Rapid heartbeat
  • Shortness of breath
  • Trembling
  • A strong desire to get away <fn></fn>

The specificity of this disorder is that it often concerns food, which is a daily need. Some foods or types of food are avoided (hence the possibility of some deficiencies, to be monitored), excessive cleanliness of the environment is often necessary for the patient (but not systematic), resulting in movements strongly influenced by phobia.

Toxophobia is similar to other phobias related to dirt, dust, diseases… except that the majority of cases the fear is about a deliberate poisoning. Patients following drug treatments, interacting with medical staff, objects such as syringes, etc., which can make it difficult to manage any associated depression and the main disorder with drug treatment.


The treatment of poison phobias is increasingly being addressed by cognitive-behavioral therapies. The efficacy of exposure therapy, in combination with other Cognitive-Behavioral Therapy (CBT) components, in the treatment of specific phobia gave good results <fn></fn>, other therapeutic trends based on a similar model (neuro-linguistic programming, virtual exposure) have also shown significant improvement.

Drugs Identification in Urine, Bile and Gastric Contents using Thin Layer Chromatography in Multiple Screening Systems

A method of simultaneous identification of 25 molecules in human urine, bile and gastric contents using liquid-liquid extraction followed by thin layer chromatography (TLC) using multiple screening systems is described. The analytes were extracted at 25°C under isocratic conditions using chloroform after acidification with 1 to 2 drops of HCl 6 N for 10 mL of the biological sample, and dichloromethane after alkalization with 1 to 2 drops of NaOH 10 N for 10 mL of the biological sample. Employing LLE, the best conditions were achieved with double extraction of 10 mL of the biological sample, pH=9.5 for alkaline extraction and pH=2 for acid extraction. The organic extractums were filtered and dehydrated using anhydrous sodium thiosulfate powder and concentrated after evaporation of the organic solvents at 65°C. The extraction residues were solubilized in 500 µl of methanol and spotted with the molecules of reference onto four TLC plates (10 cm × 10 cm). The TLC plates were put into twin-through development chambers previously incubated 30 minutes for saturation namely TA (methanol:ammoniac 5% (50:0.750, v/v)), TD (chloroform:acetone (40:10, v/v)), TE (Ethyl acetate:methanol:ammoniac (42.3:5:2.5, v/v/v)), TB (cyclohexan:toluene:diethylamine (37.5:7.5:5, v/v/v)). The mobile phase migrates by capillarity through the stationary phase, driving at different speeds the molecules to be separated. The migration time (several minutes) depends on various parameters. When the solvent front has moved through a distance considered as sufficient (a few centimetres), the TLC plates were removed and dried, then exposed to ultraviolet light, the retardation factors Rf of each visible spot was measured. Some chemical processes might also be used to reveal spots. The total number of substances present in the biological sample was determined by counting the number of spots found on each TLC plate, the biggest number among the four counted values is considered as the default number of the present substances. A mathematical formula was applied to guess all possible matches according to a data table of Rf profiles of standards already calculated by the same method. The validation parameters obtained in LLE were linearly range of 50-1000 µg mL-1 biological fluid (r≥0.9815). This method has shown its suitable applicability in order to rapidly identify a wide verity of substances of toxicological interest present in the biological samples. Moreover, it’s inexpensive and could be suggested in various routine drug screening processes, especially for toxicological/forensic analysis.

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Genotoxicity evaluation using flow cytometry based micronucleus test in HepG2 cells

Humans are exposed to the contaminated environment which may contain known human or animal carcinogens and it was estimated that environmental chemicals contribute to 7 19 % of cancers in humans (Goodson et al. 2015). Among the total number of Group 1 agents (known human carcinogens as per IARC classification), most belong to different classes of chemicals (Baan et al. 2009).

Polyaromatic hydrocarbons (PAHs) and metals are the major chemicals in the environment and have been associated with adverse health effects in humans. PAHs are formed during the incomplete combustion of organic materials and also present in cigarette smoke, vehicle exhaust, coal tar, and charcoal broiled foods. In 1775, Sir Percival Pott first observed an association between an increased incidence of scrotal cancer in chimney sweeps and exposure to soot (Pott 1775). Since then many epidemiological studies in PAH exposed workers have reported the increased incidence of cancers in humans (ATSDR 1995, Bosetti et al. 2007, Zhang et al. 2009, IARC 2010, Silverman et al. 2012, Kim et al. 2013).

There are no epidemiological data available for incidences of cancer followed by B[a]P exposure alone. But studies with experimental animals showed that B[a]P produced tumors in all tested species by different routes of exposure (IARC 2012a) Occupational exposure to B[a]P containing PAHs mixtures have been associated with increased risk of cancer (lung, bladder, skin and hematolymphatic system IARC 2010). Arsenic (As) and cadmium (Cd are known human carcinogens. Occupational and environmental exposure to Cd causes lung, kidney, and prostate cancers (IARC 2012b). Arsenic exposure has been linked with lung, urinary bladder, skin and kidney cancers in humans (IARC 2012c).

Mixed contamination of PAHs, As, Cd, and Pb is elevated in the environment due to increased industrial and anthropogenic activities (Megharaj and Naidu 2008 .

Benzo[a]pyrene, As, Cd, and Pb are top priority pollutants in the ATSDR substance priority list (ATSDR 2015) and also the major contaminants in various contaminated sites around the world (Megharaj and Naidu 2008, Panagos et al. 2013). The data on the co genotoxicity of metals and B[a]P have so far been limited to binary mixtures of B[a]P with As or Cd and the co genotoxic effects of metals are inconsistent in these studies which indicate synergistic or antagonistic effect of metals on the genotoxicity of B[a]P (Maier et al. 2002, Tran et al. 2002, Mukherjee et al. 2004, Fischer et al. 2005, Lewińska et al. 2007, Simon et al. 2014). There are several hundred PAHs among which 17 PAHs are prioritized due to their common occurrence in the contaminated sites and potential for human exposure (ATSDR 1995).

Simple hydrocarbons like naphthalene (Nap), phenanthrene (Phe) and pyrene (Pyr) are common non carcinogenic PAHs found along with B[a]P in most of the contaminated sites (ATSDR 1995). Pyrene is one of the abundant hydrocarbons in PAHs mixtures and used as a biomarker of PAH exposure (Silins and Högberg 2011). Naphthalene is classified as Group 2B, Phenanthrene and Pyr are Group 3 carcinogen (IARC, 2010). The genotoxicity of PAHs mixtures or as a binary combination of B[a]P with other PAHs vary depending on the PAHs that are present in the mixtures and the reported findings include synergism, additivity or antagonism (Staal et al. 2008, Tarantini et al. 2009, Jarvis et al. 2013).

The In vitro micronucleus test has been routinely used to screen the genotoxicity of pharmaceutical agents and environmental chemicals. This assay has higher throughput than the traditional genotoxicity assays and reliable MN measurement help to determine the genotoxicity of chemicals (Bryce et al. 2007).

In this study, HepG2 cells are used as a test system, since liver plays a major role in the metabolism of PAHs and is one of the target organs of metal toxicity. Apart from the inherent capacity of metabolic enzymes, these cells express p53 protein which will be useful to screen the p53 mediated DNA damage (Boehme et al. 2010, Zager et al. 2010) In our previous study, we have reported the genotoxicity of mixtures of As, Cd and PAHs in HepG2 cells (Peng et al. 2015). But this study evaluated only the binary mixtures of metals (As and Cd) with B[a]P and a quaternary combination of B[a]P with Nap, Phe, and Pyr.

This study did not evaluate the genotoxicity of all possible mixture combinations of metals and PAHs. In addition, the MN was counted manually which was low in throughput. In this study, a flow cytometry based MN test was used to determine the individual and combined effects of As, Cd, Pb and PAHs (Nap, Phe and Pyr) on the genotoxicity of B[a]P in HepG2 cells.

The genotoxic effects were determined for the binary, ternary, quaternary and seven component mixtures of PAHs and metals. In addition, the individual and combined effects of Cd and Pb on the genotoxicity of As was determined. The AhR pathway plays a major role in the toxicity of PAHs (Baird et al. 2005, Galván et al. 2005, Nebert and Dalton 2006). In this study, the effects of As, Cd, Pb and PAHs (Nap, Phe and Pyr) on the AhR activation of B[a]P were determined using the HepG2 cell based chemically activated fluorescent (CAFLUX) assay.

Materials and methods


Cell culture medium DMEM (Dulbecco’s Modified Eagle Medium), Trypsin EDTA (0.25%), penicillin streptomycin solution and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies Ltd, VIC, Australia). CellTiter 96 Aqueous One Solution Cell Proliferation Assay (G3581) was purchased from Promega Corporation, Madison, USA.

The In Vitro microflow kit was purchased from Litron aboratories Ltd, Rochester, NY, USA. The kit contains incomplete lysis solutions 1 and 2, nucleic acid dye A (ethidium monoazode, EMA), nucleic acid dye B (SYTOX® green nucleic acid stain), RNase solution and 10X buffer. PeakFlowTM green flow cytometry reference beads, 6 µm was purchased from olecular robes (Life Technologies Ltd), USA. Benzo[a]pyrene (B[a]P), (CAS number: 50 32 8), naphthalene (CAS number: 91 20 3), phenanthrene (CAS number: 85 01 8), pyrene (CAS number: 129 00 0), cadmium chloride (CAS number: 10108 64 2), lead acetate (CAS number: 6080 56 4) and sodium arsenite (CAS number: 7784 46 5) were purchased from Sigma Aldrich (St. Louis, MO, USA)

Cell culture

HepG2 cells (ATCC No. HB 8065) were obtained from ATCC (American type culture collection), Manassas, USA.

The cells were maintained as a subconfluent monolayer in 75 cm culture flask using the modified DMEM medium with 10% (v/v) FBS + penicillin streptomycin (50 Units/mL) and used for experiments after two weeks of thawing from cryopreserved stock. The cells were harvested using 0.25% Trypsin EDTA (prewarmed to 37ºC).

For cytotoxicity and MN study, the cells (1×10 cells/well) were seeded into 96 well plates and incubated at 37 °C under 5% CO in an incubator (Heracell 150i CO incubator, Thermo Scientific, Australia) for 24 h before chemical treatment. Stock solutions of PAHs (B[a]P, Nap, Phe and Pyr) and metals (As, Cd and Pb) were prepared in DMSO and MilliQ water (18 MΩ.cm, Merck Millipore, VIC, Australia), respectively. The working solutions prepared in DMEM + FBS (10%) medium or DMEM alone were used for MN test and Ah CAFLUX assay, respectively.

The working solutions were added to the plates reaching a final concentration of 0.5% v/v of vehicle control (DMSO or MilliQ water).

In vitro flow cytometry micronucleus test

The selected concentrations of individual MN study were 0 to 2 μM for B[a]P and Cd; 0 to 10 μM for As and 0 to 100 μM for Pb. Initially, the cytotoxicity of PAHs and metals were determined using the MTS (tetrazolium compound [3 (4,5 dimethylthiazol yl) (3 carboxymethoxyphenyl) (4 sulfophenyl) 2H tetrazolium]) assay. The cytotoxicity assay was carried out as described by Muthusamy et al. (2016b).

Based on their cytotoxicity to HepG2 cells, the concentrations of metals and PAHs were selected for the MN study. The treatment was carried out for 1.5 – 2 normal doubling period of HepG2 cells (OECD 2014). The details of selected individual concentrations are provided in Table 1. Cell staining and lysis were carried out as instructed by the manufacturer, Litron Laboratories Ltd, Rochester, NY, USA. In brief, the plates were removed from the CO incubator after a treatment period of 48 50 h and placed on ice for 20 min.

Table 1 . Concentrations of individual and mixtures of PAHs, As, Cd, and Pb of genotoxicity evaluation using flow 2 cytometry based micronucleus (MN) test in HepG2 cells.

The supernatant was carefully removed and nucleic acid dye A working solution (EMA + 1 x buffer solution) was added to each well. Then, the plates were exposed to light for photoactivation and the cells were washed with 1 x buffer solution. The complete lysis I solution (containing lysis solution + Sytox Green + RNase) was added to the cells and incubated at 37ºC for 1 h. Then, lysis II solution (lysis II + Sytox green + counting beads) was added, and the plates were further incubated at room temperature for 30 min and proceeded to flow cytometry analysis.

Flow Cytometry analysis

Flow cytometry analysis of MN was carried out using the BD LSR II analyser (Becton, Dickinson, and Company, BD Biosciences, San Jose, USA). The xperimental template provided by Litron Laboratories was used for analysis. The cells were re suspended and protected from light during the analysis. Initially, vehicle control samples were used to adjust the voltage to capture the nuclei and an average 7,500 10,000 events/well were recorded.

The data analysis was carried out using BD FACSDiva TM Software (BD FACSDiva TM Software Reference Manual, Version 8.0.1, BD Biosciences, San Jose, USA). The MN values were expressed as frequency percent and calculated by dividing the number of events that fall within the “MN” region by the number of events that fall within the “Nucleated” region and multiplying by 100. The criterion to consider the positive response is that increase of greater than two fold in mean % MN in the individual or chemical mixtures treated groups compared to concurrent vehicle control values (Shi et al. 2010).

Mixture experiments- Determination of effects of multi-component mixtures of PAHs and metals on MN formation

The individual concentration response of the MN test showed that B[a]P and As gave a clear positive response for MN formation. The mixture experiments were designed to study the effects of individual or mixtures of As, Cd, and Pb/or PAHs on the genotoxicity of B[a]P. In this study, two independent experiments were conducted for binary, ternary, quaternary, and seven component mixtures of PAHs and metals.

In this study, the experiments for binary, ternary, quaternary, and seven component mixtures of PAHs and metals were carried out in two steps. In step one, the experiments were conducted using the binary combinations of PAHs and metals. Based on results obtained from step 1, the next set of studies was extended to ternary to seven chemical mixtures combinations. In addition, the effects of Cd and Pb on the MN formation of As were also determined. The results of individual and different combinations of PAHs and metals are expressed as fold change in the MN frequency compared to the vehicle control. The details of selected concentrations of the mixture study are provided in Table 1. The cytotoxicity of the selected concentrations of the PAHs and metal mixtures were determined using the MTS assay.

Cell cycle analysis

The cell cycle parameters, G1, S and G2/M were analyzed using ModFit LT (version 3.2) Software (ModFit LT user guide), Verity Software House, ME, USA. The linear scale recording of nucleus fluorescence intensity from flow cytometry analysis was used for cell cycle analysis.

Determination of activation of AhR using HepG2 cell based

CAFLUX assay HepG2 cells transfected with the reporter plasmid, pGreen1.1 (dioxin responsive enhanced green fluorescent protein, EGFP) was used to determine the effects of individual or mixtures of B[a]P with metals or PAHs on the AhR. In our previous study, we have reported the procedure for transfection of HepG2 cells with the reporter plasmid, pGreen1.1 (Peng et al. 2015) Cell culture: The cells were maintained in a medium containing DMEM + 10% (v/v) FBS + G418 sulphate + penicillin streptomycin (50 Units/ml). On the day of experiments, cells (30,000 cells/well) were seeded into 96 well plate (Corning® 96 well flat clear bottom, sterile black polystyrene TC treated microplates, Corning Life Sciences, NY, USA) and incubated at 37 °C under 5% C 2 for 24 h. The individual dose response study of AhR activation was carried out for B[a]P, Nap, Phe, and Pyr.

For mixtures studies, the effects of metals (Cd (0 to1 µM), As (0 to10 µM) and Pb (0 to 50 µM)) and other PAHs ((Nap, Phe and Pyr, 0 to 15 µM) on the AhR activity of B[a]P (2 µM) was determined. After the chemical treatment, the cells were incubated at 33ºC under 5% CO for 72 h. It was observed that cells that are grown at 33 ºC results in several fold greater EGFP activity than cells incubated at 37 ºC (Zhao et al. 2010). After the treatment period of 72 h, the fluorescence intensity as a measurement of EGFP expression was recorded using FLUOstar Omega, BMG Labtech, VIC, Australia.

The fluorescence intensity of control and treated groups was presented after deduction of background fluorescence intensity.

Statistical analysis

The data from MN test, cell cycle analysis, and the AhR activation assay were analysed by “one way ANOVA followed by Turkey’s multiple comparison tests using GraphPad Prism version 6.00 for Windows”, GraphPad Software, La Jolla California USA, ( In case of MN test, the fold change in the MN frequency compared to the vehicle control were calculated for individual and chemical mixtures. The results of fold change in the MN frequency was used for statistical analysis and the fold change of MN frequency from chemical mixture treated group were compared against B[a]P or As depending on the type of chemical mixtures. he significant difference between the control and treated groups was evaluated at p <0.05.



The cytotoxicity of binary, ternary, quaternary and seven component mixtures of PAHs, As, Cd, and Pb are presented in Supplementary Material Figure S1. At the selected concentrations, the individual PAHs and metals were not toxic to HepG2 cells. The binary to multi component mixtures of PAHs, As, Cd, and Pb were found to be toxic to HepG2 cells (up to 40% reduction in cell viability). In the case of B[a]P and metals mixtures, the binary mixture of B[a]P + As and the ternary mixture of B[a]P + Cd + Pb caused 38 and 35% of reduction in cell viability, respectively. Among the B[a]P + PAHs mixtures, a maximum reduction of 39% in cell viability was observed with B[a]P + Phe and B[a]P + Nap + Phe mixtures. The quaternary mixtures of B[a]P with metals (26%) or PAHs (25%) and multi component mixtures of PAHs and metals (34%) also reduced the cell viability of HepG2 cells.

Micronucleus test

Benzo[a]pyren was clearly positive for MN formation in HepG2 cells but other PAHs (Nap, Phe and Pyr) did not elicit positive responses. Among the metals, As was clearly positive for MN formation, and Cd and Pb showed a weak positive response effect for MN formation. The effects of individual PAHs and metals on MN formation in HepG2 cells are presented in Supplementary Material Figure S2.

Effects of individual and mixtures of metals on the genotoxicity of B[a]P

The binary mixture studies were conducted for mixtures of B[a]P (2 µM) with As (5 and 10 µM) or Cd (0.3 and 1 µM) or Pb (25 and 50 µM) (Figure 1). The binary mixtures of B[a]P with metals significantly increased the MN formation compared to B[a]P alone. At the selected low and high concentrations of mixtures the binary combinations of B[a]P and metals resulted in two and four fold increase of MN formation compared to B[a]P alone, respectively.

Figure 1. Effects of mixtures of B[a]P and metals on micronucleus (MN) formation in HepG2 cells. Results are expressed as fold change in MN formation compared to vehicle control. * indicates significant difference (p<0.0 ) compared to B[a]P alone. B[a]P benzo[a]pyrene, As arsenic, Cd cadmium and Pb lead. Values are mean ± SD, n= 4.

The ternary mixture studies were conducted using B[a]P (2 µM) with As + Cd (2.5 + 0.15 and 5 + 0.5 µM) or As + Pb (2.5 + 12.5 and 5 + 25 µM) or Cd + Pb (0.15 + 12.5 and 0.5 + 25 µM) (Figure 1). The ternary mixtures of B[a]P and metals increased the MN formation compared to B[a]P alone. Among the ternary mixtures, the mixture of B[a]P + As + Cd (2 + 5 + 0.50 µM) showed a maximum increase of 4.7 fold in the MN formation compared to B[a]P alone. The quaternary mixture of B[a]P + As + Cd + Pb did not show a significant increase in the MN formation (2 fold vs B[a]P alone) compared to B[a]P alone (Figure 1).

Effects of individual and mixtures of Nap, Phe and Pyr on the genotoxicity of B[a]P

The binary combination of B[a]P with Nap or Phe or Pyr and ternary mixtures (B[a]P + Nap + Phe, B[a]P + Nap + Pyr and B[a]P + Phe + Pyr) decreased the MN formation compared to B[a]P alone (up to 60% reduction in MN formation Vs B[a]P alone) (Figure 2).

Figure 2. Effects of multi component mixture of PAHs and metals and mixtures of metal on micronucleus (MN) formation in HepG2 cells. Results are expressed as fold change in MN formation compared to vehicle control. * indicates significant difference (p<0.0 ) compared to B[a]P or As alone. B[a]P benzo[a]pyrene, As arsenic, Cd cadmium, Nap naphthalene, Pb lead, Phe phenanthrene and Pyr pyrene Values are mean ± SD, n= 4.

The quaternary mixture (B[a]P + Nap + Phe + Pyr) and seven component mixtures (containing both PAHs and metals) did not show significant difference in the MN formation compared to B[a]P alone (Figure 2). 3.1.3 Effects of Cd and Pb on the genotoxicity of As The binary and ternary mixtures of Cd and Pb with As increased the MN formation compared to As alone (Figure 2). Among the binary mixtures, As + Pb mixture showed a maximum of 2 fold increase in the MN formation compared to As alone. In the case of a ternary mixture of As + Cd + Pb, the observed increase of MN formation was less than those of binary mixtures (Figure 2).

Effects of individual and mixtures of PAHs and metals on cell cycle parameters

The effects of mixtures of PAHs and metals on cell cycle distribution and percentage of cells in different phases G1, S and G2/M in HepG2 cells are presented in Tables 1 to 2. The individual treatment of B[a]P alone reduced the cell population in G1 phase and increased the cell accumulation in G2/M phase compared to vehicle control, respectively. The binary combination of B[a]P with metals decreased the cell population in G1 phase (maximum of 39%) and mixtures of B[a]P with As or Cd (at higher concentration combination) increased the cell accumulation in G2/M phase (maximum of 60%) compared to B[a]P, respectively (Table 1).

In the case of ternary mixtures, B[a]P + As + Cd decreased the cell population in G1 phase and increased the cell accumulation in G2/M phase, respectively. The other ternary mixtures of B[a]P and metals (B[a]P + As + Pb and B[a]P + Cd + Pb) decreased the cell numbers in G1 phase and increased the cell population in S phase, respectively. The treatment related changes of increase and decrease of the cell population in G1 and G2/M phase ere observed with a quaternary mixture of B[a]P + As + Cd + Pb, respectively (Table 1). The mixtures of B[a]P with Nap, Phe and Pyr did not cause any changes in the cell cycle parameters (G1, S and G2/M phases) compared to B[a]P alone (Table 2).

Similarly, quaternary and seven component mixtures did not result in any changes in cell cycle parameters compared to B[a]P alone. The metal mixtures, As with Cd or Pb (binary combination of As + Pb (10 +50 ), As + Cd (10 +1 µM) and As +Cd + Pb (10 +25 + 0.5 µM) decreased the cell population in G1 hase and increased the cell accumulation at G2/M phase, respectively (Table 2).

Table 2: Effects of mixtures of B[a]P and metals on cell cycle parameters in HepG2 cells

Effects of individual PAHs on activation of AhR

The individual PAHs activated the AhR in HepG2 cell based CAFLUX assay. Among the PAHs, B[a]P was a potent inducer of the AhR. The remaining three PAHs (Nap, Phe and Pyr) also activated the AhR, but the observed effect were less than that of B[a]P (Supplementary Material Figure S3).

Effects of individual and mixtures of B[a]P and metals on activation of AhR

Individual (As or Cd or Pb) or combinations (As + Cd or As + Pb or Cd + Pb) of metals did not activate the AhR in HepG2 CAFLUX assay. The binary (B[a]P + As or Cd or Pb) or ternary mixtures (B[a]P + (As + Cd) or (As + Pb) or (Cd + Pb) of B[a]P and metals significantly increased the activation of AhR compared to B[a]P alone (Figure 3). Among these mixtures, the mixture of B[a]P + As and B[a]P + As + Pb resulted in a maximum of 17% increase in the AhR activity compared to B[a]P alone.

Figure 3. Effect of arsenic (As), cadmium (Cd) and lead (Pb) on AhR activation of B[a]P in HepG2 cell based CAFLUX (chemical activated fluorescence gene expression) assay. The changes in the expression of enhanced green fluorescent protein (EGFP) indicate activation of AhR and measured as fluorescence intensity (FI). * indicates significance (p<0.0 ) compared to B[a]P. Values are mean ± SD, n= 3.

Effects of individual and mixtures of B[a]P, Nap, Phe, and Pyr on activation of AhR

The binary combination of B[a]P with Nap, Phe and Pyr (B[a]P + Nap or Phe or Pyr) or ternary combinations (B[a]P + (Nap + Phe) or (Nap + Pyr) or (Phe + Pyr) reduced the activation of AhR compared to B[a]P alone (Figure 4). The mixtures of B[a]P + Phe and B[a]P Nap + Phe resulted in 16 and 12% reduction in the AhR activity compared to B[a]P alone, respectively.

Figure 3. Effects of individual and mixtures of naphthalene (Nap), phenanthrene (Phe) and pyrene (Pyr) on AhR activation of B[a]P in HepG2 cell based CAFLUX (chemical activated fluorescence gene expression) assay The changes in the expression of enhanced green fluorescent protein (EGFP) indicate activation of AhR and measured as fluorescence intensity (FI). * indicates significance (p<0.0 ) compared to B[a]P. Values are mean ± SD, n= 3.


Measuring Apoptosis by Flow Cytometry

Mitochondria are double-membraned organelles believed to have been integrated into modern eukaryotes via symbiosis of proteobacteria into an anaerobic pre-eukaryotic (host) cell 1.5–2 billion years ago. According to modern thinking (pioneered by Mitchell;), an essential role of mitochondria is to produce ATP via oxidative phosphorylation (OXPHOS). In this process, the chemical energy stored in nutrients (carbohydrates, fats, etc.) is converted to an electrochemical gradient across the inner mitochondrial membrane via the electron transport chain (ETC) complexes. This electrochemical gradient acts as a store of energy. ATP synthase uses this stored energy to convert ADP to ATP. This bioenergetic picture of the role of mitochondria is now widely accepted. A second role of mitochondria is in the so-called intrinsic apoptosis pathway. This pathway converges (figuratively and literally) at the membrane of the mitochondria. Upon certain cell death signals [such as reactive oxygen species (ROS), DNA damage, etc.], the outer membrane of mitochondria becomes permeable enough to release the soluble hemeprotein cytochrome C (CytC), as well as Smac/Diablo, endonuclease G, and other intermembrane space proteins, which irreversibly activate downstream caspases to carry out the apoptosis process.

Apoptosis is a programmed mode of cell death that is accompanied by numerous morphological as well as biochemical changes to the cellular architecture. This results not only in cell death but also in the effi- cient removal of apoptotic cells by phagocytes. Apoptotic cells display a range of common characteristics that include cell shrinkage, plasma membrane blebbing, cell detachment, nuclear condensation, DNA fragmentation, externalization of phosphatidylserine (PS) and activation of caspases. In contrast, necrotic cell death is characterised by rapid plasma membrane, organelle swelling and plasma membrane rupture with none of the features of apoptosis. Apart from severe physical stresses, necrotic cell death often betrays the activities of viral infection and the activities of bacterial toxins. While necrotic cell death is characterized by the release of endogenous ‘danger signals’ and subsequent inflammation, apoptosis is largely tolergenic. Therefore, care must be taken when assessing whether cells are dying via apoptosis or necrosis. Here, we highlight a number of assays, utilizing flow cytometry, to determine whether cells have undergone apoptosis or alternative modes of cell death.

Detection of fragmented DNA by flow cytometry as a measure of apoptotic cell death

Intranucleosomal DNA fragmentation is a major hallmark of apoptosis. DNA fragmentation may be assessed by flow cytometry. Analysis of a cell population’s replication state (cell cycle profile) can be readily achieved with the fluorescent dye Propidium iodide (PI), which binds stoichiometrically to nucleic acids resulting in a fluorescence emission proportional to the DNA content of the cell. The rationale behind the approach is as follows: quiescent and G1 cells have two chromosome copies, while cells undergoing mitosis G2/M have double the amount of DNA and so will have double the fluorescence intensity of G1 cells. Cells in S phase will have a fluorescent signal between G1 and G2/M, because these cells are synthesizing DNA on their way to G2/M (Fig. 3A).

Figure 3. Measure of DNA fragmentation during apoptosis by flow cytometry. Jurkat cells were treated with 200 ng/ml anti-Fas (CH-11). Cells were harvested at indicated timepoints (A–E) and analysed by flow cytometry. (F) Gating strategy to discriminate cells aggregates from single cells.

Due to the generation of lowmolecular weight DNA fragments during apoptosis, cells undergoing apoptosis can be readily identified on DNA content histograms as cells with fractional hypodiploid or ‘‘sub-G1’’ content (Fig. 3B–E). Cellular DNA content is measured using a fluorescent dye after cell fixation with ethanol. Cell fixation does not retain small nuclear fragments in apoptotic cells. These low molecular weight DNA fragments leak out during subsequent wash steps. As a result, apoptotic cells contain a fractional DNA content relative to viable cells that can be readily distinguished by flow cytometry.


The following protocol is tailored towards suspension cells, however, if using adherent cells remember to harvest the supernatant (late apoptotic cells become detached and float in the medium) in addition to the adherent/semi adherent cells on the plate and proceed as outlined below:

1.Apoptosis was induced in 2 10^6 Jurkat cells by incubation with 200 ng/ml anti-Fas antibody (CH-11) for 1–4 h. Cells are harvested at the desired time points and centrifuged at 400g for 5 min. Cells are washed with PBS pH 7.2 and centrifuged at 400g for 1 min.

2.Resuspend cells in 1 ml ice-cold 70% ethanol and incubate for at least 1 h at -20 °C to fix (cells can be stored for up to 6 months at -20 °C).

3.Centrifuge cells at 2500g for 5 min (a higher centrifuge speed is required as fixed cells become buoyant and may fail to pellet or stick along the side of the eppendorf). Aspirate off the ethanol without disturbing the cell pellet and resuspend with 1 ml phosphate-citrate wash buffer (200 mM Na2HPO4, 100 mM citric acid) followed by centrifugation at 2500g for 1 min.

4.To stain nuclei, prepare PBS pH 7.2 containing propidium iodide 10 lg/ml and RNase A 100 lg/ml (included to degrade RNA and to prevent PI staining of RNA) and incubate with cells for 30 min.

5.Samples are ready for analysis by flow cytometry (no need to wash out PI/RNase but this can be done if desired).

Setting correct parameters for cell cycle analysis by flow cytometry

A few considerations must be taken into account when using flow cytometry for cell cycle analysis:

A) Ensure that the fluorescence channel 2 (FL2) is set at linear (LIN) scale. It is harder to distinguish the differential fluorescence between G0/G1 and G2/M peaks on a logarithmic (LOG) scale. LIN amplification allows for clear separation between G0/G1 and G2/M peaks.

B) A common problem to control for during cell cycle analysis is aggregation of cells. For example cells can stick together and pass through the flow cytometer’s laser intercept simultaneously. In either case, two cells in G0/G1 that are stuck together or pass through the laser intercept at the same time will have a fluorescence signature equivalent to a cell in G2/ M. Therefore, the number of events recorded as G2/M will be artificially high. A way to exclude these events is by excluding non-single cell events from the analysis using scatter properties (FSC/SSC).

C) To discriminate between cellular aggregation and single cells, select a plot with FL2-A parameter as the y-axis and FL2-W as the x-axis (Fig. 3F). Single cells (G0/G1 or G2/M) will have pulse width values (FL2-W) that are similar, however aggregates will have larger pulse width values (due to increased cell width). In the example (Fig. 3F), single cells have been gated (R1-single cells) and the FL2-A histograms (Fig. 3A–E) have been formatted to display only events within this region (R1-single-cells).

Reference : ScienceDirect Measuring apoptosis by microscopy and flow cytometry