**3. Cytotoxic effects of nisin on non-malignant mammalian cells**

It is clear that nisin is an effective antimicrobial agent which can inhibit the growth of/kill several Gram-positive bacterial species, including food-borne pathogens such as *Staphylococcus aureus*, *Listeria monocytogenes* and *Clostridium botulinum* as well as exhibiting activity against many clinical important pathogens such as vancomycin-resistant *Enterococci* (VRE), *Streptococcus pneumonia* and MRSA [32, 47, 48]. Despite having exceptional antimicrobial activity, many AMPs also exhibit high toxicity to mammalian cells. An example of a cytotoxic AMP is melittin, the main active component of apitoxin (bee venom). Melittin has an excellent antibacterial activity and the antimicrobial mechanism of this AMP is most likely the permeabilisation of cell membranes by pore formation resulting in cell lysis and death [49]. Although melittin has effective broad-spectrum antimicrobial activity, this AMP is extremely toxic to mammalian cells even at very low concentrations.

As mentioned before, nisin has a *Generally Regarded as Safe* (GRAS) status and is considered safe for human consumption. The Accepted Daily Intake (ADI) of nisin was determined by the FDA as 2.94 mg/per day (0.049 mg/kg body weight/day) in 1988, prior to receiving GRAS status [50]. In a study by Joo and co-workers, mice were exposed to a concentration of nisin more than ×1000 (150 mg/kg body weight/day) the recommended ADI over a period of 3 weeks with no signs of cytotoxicity [51]. In another study, mice were treated with doses of 800 mg/kg body weight/day (more than 10 000 times higher than the recommended ADI) ultra-pure nisin Z for 3 weeks without any evidence of toxicity [52]. In both these studies, long-term (>3 weeks) treatment with high concentrations of nisin did not result in any observable toxicity.

We also investigated cytotoxicity of nisin Z towards mammalian cells using the MTT assay to measure metabolic activity and the lactate dehydrogenase (LDH) assay to indicate membrane integrity. The non-malignant human immortalised keratinocyte (HaCaT) cells were employed for cytotoxicity testing and cultured under normal conditions [24]. Briefly, HaCat cells were seeded in a 96-well plate and incubated until ~90% confluent. Synthetic melittin was used (≥97% HPLC from Sigma-Aldrich) as a positive AMP control for cytotoxicity. After 24 h of exposure to nisin Z or melittin (2.5–40 μg/ml), the MTT assay was performed as described previously [24]. The ability of NAD(P)H-dependent cellular oxidoreductase enzymes to reduce MTT to formazan is considered a reflection of the number of viable cells present. Cell viability is expressed as a percentage relative to the untreated control, which was set as being 100% viable. For an assay positive control, cells were exposed to 0.01% Triton-X 100 (Sigma-Aldrich, St Louis, MO, USA).

Furthermore, *S. epidermidis* treated with ampicillin-nisin Z combination also showed an additive interaction. Novobiocin-nisin Z combinations showed synergistic interactions when used against *S. epidermidis* and *S. aureus.* Novobiocin, as part of the aminocoumarins antibiotic group, is able to indirectly block DNA replication by effectively inhibiting bacterial DNA gyrase. Novobiocin-nisin Z combination was particularly effective in the treatment of *S. aureus* as a dramatic reduction in the ƩFIC was witnessed. This may be due to the different, but complementary, mechanisms of actions of nisin Z and novobiocin. As the lipid II-nisin Z complex forms pores in the bacterial membrane, hydrophobic novobiocin can pass through the cell membrane to interact with the DNA gyrase of *S. aureus*. This is only speculation and the exact synergistic mechanism of should be examined further. This *in vitro* study shows the potential of nisin Z for the use as an adjuvant with conventional antibiotics. AMPantibiotic combination therapy may aid in reinforcing the defences against resistant organisms by making it more challenging for a bacterial strain to adapt to multiple antimicrobial mechanisms. Furthermore, novobiocin is used for the treatment of mastitis in lactating cows [46]; and as previously mentioned, some nisin-based products have been developed for the treatment of mastitis. The synergistic interactions between nisin and novobiocin make this combination especially of interest for developing novel formulations for the treatment of

**3. Cytotoxic effects of nisin on non-malignant mammalian cells**

mammalian cells even at very low concentrations.

result in any observable toxicity.

It is clear that nisin is an effective antimicrobial agent which can inhibit the growth of/kill several Gram-positive bacterial species, including food-borne pathogens such as *Staphylococcus aureus*, *Listeria monocytogenes* and *Clostridium botulinum* as well as exhibiting activity against many clinical important pathogens such as vancomycin-resistant *Enterococci* (VRE), *Streptococcus pneumonia* and MRSA [32, 47, 48]. Despite having exceptional antimicrobial activity, many AMPs also exhibit high toxicity to mammalian cells. An example of a cytotoxic AMP is melittin, the main active component of apitoxin (bee venom). Melittin has an excellent antibacterial activity and the antimicrobial mechanism of this AMP is most likely the permeabilisation of cell membranes by pore formation resulting in cell lysis and death [49]. Although melittin has effective broad-spectrum antimicrobial activity, this AMP is extremely toxic to

As mentioned before, nisin has a *Generally Regarded as Safe* (GRAS) status and is considered safe for human consumption. The Accepted Daily Intake (ADI) of nisin was determined by the FDA as 2.94 mg/per day (0.049 mg/kg body weight/day) in 1988, prior to receiving GRAS status [50]. In a study by Joo and co-workers, mice were exposed to a concentration of nisin more than ×1000 (150 mg/kg body weight/day) the recommended ADI over a period of 3 weeks with no signs of cytotoxicity [51]. In another study, mice were treated with doses of 800 mg/kg body weight/day (more than 10 000 times higher than the recommended ADI) ultra-pure nisin Z for 3 weeks without any evidence of toxicity [52]. In both these studies, long-term (>3 weeks) treatment with high concentrations of nisin did not

mastitis.

26 Cytotoxicity

To investigate the effect of the two AMPs on cell membrane integrity, the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, Madison, WO, USA) was employed. This assay determines the release of lactate dehydrogenase (LDH) into the culture media from cells with impaired cell membranes. HaCat cells were exposed to melittin and nisin Z as described earlier. A lysis solution (Promega) was used as a maximum LDH release positive control. The LDH release assay was performed as described previously [24]. Results are conveyed relative to the untreated control (set to 0% LDH release) and the maximum release sample (set to 100% LDH release).

Cytotoxicity data (**Figure 2**) shows that nisin Z did not negatively affect the cell viability of HaCat cells.

The MTT assay indicates that the ability of NAD(P)H-dependent cellular oxidoreductase enzymes to reduce MTT to formazan was not affected by the exposure to the tested nisin

**Figure 2.** Cytotoxicity assay of HaCat cells exposed to the AMPs melittin and nisin Z. (A) MTT assay and (B) LDH release assay. Vehicle control groups are represented by 0 mg/ml. Values represent mean stdev n = 3. \*\*\*p < 0.001 compared to the vehicle control group.

Z concentrations. Indicating that nisin Z did not negatively affect the cell viability of HaCat cells. The LDH assay also showed that there was no significant increase in the release of LDH, indicating that nisin Z did not cause any measurable membrane damage. On the other hand, both the MTT and LDH assays showed that relatively low concentrations of melittin led to a considerable increase in cytotoxicity in HaCat cells.

Contrary to most types of cancer, the frequency of melanoma has been increasing over the last three decades [54]. In addition to a high mortality rate, Melanoma cells also have a sinister tendency to rapidly develop resistance to mainstream chemotherapeutic agents [12, 13]. *In vitro* cytotoxicity of the nisin Z was determined by employing the MTT assay, LDH assay and flow cytometric apoptosis and necrosis analyses. The non-malignant human keratinocyte (HaCat) cell line was used as a control. The MTT and LDH assays were performed, as described previously [56]. The flow cytometric FITV Annexin V apoptosis assay (BD PharmingenTM, BD Biosciences, San Jose, CA, USA) was employed for the detection of apoptotic cytotoxicity. FITC Annexin emits green fluorescence and its presentation indicates early apoptotic events, while propidium iodide (PI) emanates red fluorescence and is associated with late apoptotic

The Cytotoxic, Antimicrobial and Anticancer Properties of the Antimicrobial Peptide Nisin Z…

http://dx.doi.org/10.5772/intechopen.71927

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The quantitative colourimetric MTT assay was used to investigate the cytotoxic effect of nisin Z on cultured melanoma cells as well as non-malignant keratinocytes. There is a clear concentrationdependent decline in cell viability observed in melanoma cells exposed to nisin Z (**Figure 3A**).

**Figure 3.** Cytotoxic effects of nisin Z on melanoma (A375) cells. (A) Cell viability was determined using the MTT assay. (B) LDH release from cells following treatment with nisin Z. Keratinocytes (HaCat) were used as a non-malignant

control. \* p< 0.05 and \*\*\* p< 0.001 compared to the control groups.

or necrotic cells.

These *in vitro* results eco many of the recent *in vivo* findings, showing that nisin exposure to non-malignant cells has very little to no cytotoxic effects. Even at concentrations of nisin Z that exceeds the MICs for *S. aureus* and *S. epidermidis* (**Figure 1**) no toxicity was observed. Keeping in mind that nisin is an effective antimicrobial agent against several Gram-positive bacterial species, including clinical important and resistant pathogens, this AMP shows promising potential for clinical application.
